Enhancing immunogenicity of streptococcus pneumoniae polysaccharide-protein conjugates

ABSTRACT

The present invention provides immunogenic compositions having one or more polysaccharide-protein conjugates in which one or more polysaccharides from  Streptococcus pneumoniae  bacterial capsules are conjugated to a carrier protein in an aprotic solvent such as dimethylsulfoxide (DMSO). The present invention also provides methods for providing an enhanced immune response to a pneumococcal polysaccharide protein conjugate vaccine comprising administering to a human subject an immunogenic composition comprising polysaccharide-protein conjugates prepared in DMSO conditions.

FIELD OF INVENTION

The present invention provides immunogenic compositions comprising atleast one Streptococcus pneumoniae polysaccharide conjugated to CRM₁₉₇using reductive amination in an aprotic solvent such asdimethylsulfoxide (DMSO). The invention also provides methods forenhancing immunogenicity of immunogenic compositions having one or morepolysaccharide-protein conjugates in which one or more polysaccharidesfrom S. pneumoniae bacterial capsules are conjugated to a carrierprotein using reductive amination performed in an aprotic solvent suchas DMSO.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is a Gram-positive bacterium and the mostcommon cause of invasive bacterial disease (such as pneumonia,bacteraemia and meningitis and Otitis media) in infants and youngchildren. Pneumococcus is encapsulated with a chemically linkedpolysaccharide which confers serotype specificity. There are over 90known serotypes of pneumococci, and the capsule is the principlevirulence determinant for pneumococci, as the capsule not only protectsthe inner surface of the bacteria from complement, but is itself poorlyimmunogenic. Polysaccharides are T-cell independent antigens, and cannot be processed or presented on MHC molecules to interact with T-cells.They can however, stimulate the immune system through an alternatemechanism which involves cross-linking of surface receptors on B cells.

Children less than 2 years of age do not mount an immune response tomost polysaccharide vaccines, so it has been necessary to render thepolysaccharides immunogenic by chemical conjugation to a proteincarrier. Coupling the polysaccharide, a T-cell independent antigen, to aprotein, a T-cell dependent antigen, confers upon the polysaccharide theproperties of T cell dependency including isotype switching, affinitymaturation, and memory induction.

There are many conjugation reactions that have been employed forcovalently linking polysaccharides to proteins. Three of the morecommonly employed methods include: 1) reductive amination, wherein thealdehyde or ketone group on one component of the reaction reacts withthe amino or hydrazide group on the other component, and the C═N doublebond formed is subsequently reduced to C—N single bond by a reducingagent; 2) cyanylation conjugation, wherein the polysaccharide isactivated either by cyanogen bromide (CNBr) or by1-cyano-4-dimethylamrnoniumpyridinium tetrafluoroborate (CDAP) tointroduce a cyanate group to the hydroxyl group, which forms a covalentbond to the amino or hydrazide group upon addition of the proteincomponent; and 3) a carbodiimide reaction, wherein carbodiimideactivates the carboxyl group on one component of the conjugationreaction, and the activated carbonyl group reacts with the amino orhydrazide group on the other component. These reactions are alsofrequently employed to activate the components of the conjugate prior tothe conjugation reaction.

Reductive amination has been utilized to conjugate S. pneumoniaepolysaccharides. See, for example, U.S. Pat. No. 8,192,746, U.S. PatentApplication Publication No. 20170021006 and International PatentApplication Publication Nos. WO2011/110381 and WO2015/110941. Reductiveamination involves two steps: (1) oxidation of the antigen, and (2)reduction of the antigen and a carrier protein to form a conjugate. Thereduction step can take place in an aqueous solvent or an aproticsolvent such as DMSO. See, e.g., International Patent ApplicationPublication No. WO2016/113644.

SUMMARY OF THE INVENTION

The present invention provides immunogenic compositions comprisingpolysaccharides from one or more of S. pneumoniae serotypes 1, 2, 3, 4,5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20,21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38conjugated to a carrier protein, wherein the conjugation reactionwhereby the polysaccharide is conjugated to the carrier protein is in anaprotic solvent. In one embodiment, for compositions having identicalserotypes, one or more of the serotypes prepared in an aprotic solventhave increased immunogenicity when compared to the same one or moreserotypes prepared under aqueous conditions.

The present invention provides immunogenic compositions comprisingpolysaccharide protein conjugates prepared from one or more of S.pneumoniae serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F,14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31,34, 35A, 35B, 35F, and 38 conjugated to a carrier protein, wherein thepolysaccharide protein conjugates are made by a process comprising thestep of conjugating the polysaccharide to the carrier protein in anaprotic solvent.

The invention also provides methods of conjugating a polysaccharide fromS. pneumoniae serotype 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A,12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A,31, 34, 35A, 35B, 35F, or 38 to a carrier protein, comprising the stepof conjugating the polysaccharide to the carrier protein in an aproticsolvent.

The invention also provides methods of treating a subject with animmunogenic composition comprising one or more polysaccharides from S.pneumoniae serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F,14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31,34, 35A, 35B, 35F, or 38 conjugated to a carrier protein, wherein thepolysaccharide is conjugated to the carrier protein in an aproticsolvent.

In certain embodiments, polysaccharides from one or more of S.pneumoniae serotypes 1, 3, 4, 5, 9V, 11A, 12F, and 14 are conjugated toa carrier protein in an aprotic solvent. In certain embodiments,polysaccharides from one or more of S. pneumoniae serotypes 2, 6C, 6D,7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 20, 21, 22A, 23A, 23B, 24F, 27, 28A,31, 34, 35A, 35B, 35F, and 38 are conjugated to a carrier protein in anaprotic solvent.

In certain embodiments, polysaccharides from one or more of S.pneumoniae serotypes 3 and 18C are conjugated to a carrier protein in anaprotic solvent. In one aspect of this embodiment, polysaccharides fromS. pneumoniae serotype 3 are conjugated to a carrier protein in anaprotic solvent. In one aspect of this embodiment, polysaccharides fromS. pneumoniae serotype 18C are conjugated to a carrier protein in anaprotic solvent.

In certain embodiments, the conjugation reaction used to conjugatepolysaccharide to the carrier protein is reductive amination.

In certain embodiments, the aprotic solvent is DMSO.

In certain embodiments, the carrier protein is CRM₁₉₇.

In certain embodiments, conjugates prepared in DMSO have a lysine lossvalue greater than 5.0. In one aspect, conjugates prepared in DMSO havea lysine loss value between 7.0 and 18.0 inclusive.

In certain embodiments, the immunogenic composition further comprisespolysaccharides from one or more of S. pneumoniae serotypes 6A, 6B, 7F,10A, 15B, 19A, 19F, 22F, 23F, and 33F conjugated to a carrier protein,wherein the conjugation reaction whereby the polysaccharide isconjugated to the carrier protein is in an aprotic solvent. In certainaspects of this embodiment, the conjugation reaction is reductiveamination. In certain aspects, the aprotic solvent is DMSO. In certainaspects, the carrier protein is CRM₁₉₇. In one aspect, the immunogeniccomposition comprises polysaccharide from S. pneumoniae serotypes 6A,6B, 7F, 18C, 19A, 19F, and 23F conjugated to a carrier protein, whereinthe conjugation reaction whereby the polysaccharide from S. pneumoniaeserotypes 6A, 6B, 7F, 18C, 19A, 19F, or 23F is conjugated to the carrierprotein is in an aprotic solvent. In certain aspects, the polysaccharideis from S. pneumoniae serotypes 18C, 19A, 19F or 23F.

In certain embodiments, the immunogenic compositions of the inventionfurther comprises polysaccharides from one or more of S. pneumoniaeserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A,11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F,23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38conjugated to a carrier protein, wherein the conjugation reactionwhereby the polysaccharide from S. pneumoniae serotypes 1, 2, 3, 4, 5,6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C,16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A,31, 33F, 34, 35A, 35B, 35F, or 38 is conjugated to the carrier proteinis in an aqueous solvent. In one aspect, between 35-100% of theserotypes in the immunogenic composition are prepared using reductiveamination under DMSO conditions and the remaining polysaccharide proteinconjugates are prepared under aqueous conditions.

In one specific embodiment, the invention provides an immunogeniccomposition consisting essentially of polysaccharides from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and33F conjugated to CRM₁₉₇ polysaccharide, wherein the conjugationreaction for S. pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23Fis in DMSO conditions and the conjugation reaction for S. pneumoniaeserotypes 1, 3, 4, 5, 9V, 14, 22F and 33F is in an aqueous solvent, andoptionally further comprising about 0.2% w/v PS-20.

The present invention also provides methods for inducing a protectiveimmune response in a human subject comprising administering any of theimmunogenic compositions of the invention. In certain embodiments, thesubject is 50 years or older and/or immunocompromised. In certainembodiments, the subject is 2 years old or younger. In certainembodiments, the subject is immunocompromised.

The present invention also provides methods for providing an enhancedimmune response to a pneumococcal polysaccharide (PnPs) proteinconjugate vaccine comprising administering to a animal subject animmunogenic composition comprising polysaccharide-protein conjugatescomprising S. pneumoniae capsular polysaccharides from a first set oftwo or more pneumococcal serotypes conjugated to one or more carrierproteins, wherein the two or more of the polysaccharide-proteinconjugates from the first set are prepared using reductive aminationunder DMSO conditions. In one embodiment, said enhanced immune responseis relative to a control animal receiving an immunogenic compositionwherein one or more of the two or more polysaccharide-protein conjugatesfrom the first set are prepared using reductive amination in aqueousconditions. In one embodiment, the control animal is a mouse. In anotherembodiment, the control animal is a human. In certain embodiments, themethods employ pneumococcal polysaccharide protein conjugate vaccinewhich comprises additional polysaccharide-protein conjugates comprisingS. pneumoniae capsular polysaccharides from a second set of pneumococcalserotypes conjugated to one or more carrier proteins are prepared usingreductive amination under aqueous conditions, wherein serotypes from thesecond set are different from serotypes in the first set.

In certain embodiments, the methods employ pneumococcal polysaccharideprotein conjugate vaccine where the pneumococcal serotypes are selectedfrom serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V,10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A,22F, 23A, 23B, 23F, 24F, 27, 28A, 33F, 34, 35A, 35B, 35F, and 38.

In certain embodiments, the methods employ pneumococcal polysaccharideprotein conjugate vaccine where polysaccharide-protein conjugates fromserotype 3 or 18C are prepared using reductive amination under DMSOconditions.

In certain embodiments, the methods employ pneumococcal polysaccharideprotein conjugate vaccine where the polysaccharide-protein conjugatesfrom the first set of pneumococcal serotypes are selected from serotypes6A, 6B, 7F, 18C, 19A, 19F, and 23F.

In certain embodiments, the methods employ pneumococcal polysaccharideprotein conjugate vaccine where polysaccharide-protein conjugates fromthe first set of pneumococcal serotypes comprise serotypes 6A, 6B, 7F,18C, 19A, 19F, and 23F, which are prepared using reductive aminationunder DMSO conditions, and polysaccharide-protein conjugates from asecond set of serotypes are prepared under aqueous conditions.

In one specific embodiment, the methods employ pneumococcalpolysaccharide protein conjugate vaccine where polysaccharide-proteinconjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F areprepared using reductive amination under DMSO conditions andpolysaccharide protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22Fand 33F are prepared under aqueous conditions.

In certain embodiments, the methods employ pneumococcal polysaccharideprotein conjugate vaccine where polysaccharide protein conjugates frombetween 35-100% of the serotypes are prepared using reductive aminationunder DMSO conditions and the remaining polysaccharide proteinconjugates are prepared under aqueous conditions. In one aspect,polysaccharide protein conjugates from between 45-80% of the serotypesare prepared using reductive amination under DMSO conditions and theremaining polysaccharide protein conjugates are prepared under aqueousconditions. In another aspect, polysaccharide protein conjugates frombetween 75-100% of the serotypes are prepared using reductive aminationunder DMSO conditions and the remaining polysaccharide proteinconjugates are prepared under aqueous conditions.

In certain embodiments, the methods employ pneumococcal polysaccharideprotein conjugate vaccine where the carrier protein is selected from thegroup consisting of Neisseria meningitides Outer Membrane ProteinComplex (OMPC), tetanus toxoid, diphtheria toxoid, protein D and CRM₁₉₇.In one aspect, the carrier protein is CRM₁₉₇.

In certain embodiments, the methods employ pneumococcal polysaccharideprotein conjugate vaccine where the conjugates prepared using reductiveamination under DMSO conditions have a higher proportion of glycopeptidebonds formed as measured by protein lysine loss value greater than 5.0.In one aspect of this embodiment, the conjugates prepared usingreductive amination under DMSO conditions have a lysine loss valuebetween 7.0 to 18 inclusive. In another aspect, the conjugates preparedusing reductive amination under DMSO conditions have a lysine loss valuegreater than 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or 10.0.

In another specific embodiment, the invention provides a method forproviding an enhanced immune response to a pneumococcal polysaccharide(PnPs) protein conjugate vaccine consisting essentially ofpolysaccharides from S. pneumonia serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM₁₉₇ polysaccharide,wherein the method comprises administering to a human subject animmunogenic composition comprising polysaccharide-protein conjugatesfrom a first set and a second set of pneumococcal serotypes, wherein thefirst set of serotypes consists of 6A, 6B, 7F, 18C, 19A, 19F, and 23Fand are prepared using reductive amination under DMSO conditions, andthe second set of serotypes consists of 1, 3, 4, 5, 9V, 14, 22F and 33Fand are prepared under aqueous conditions.

In certain embodiments, the enhanced immune response in animalsvaccinated with immunogenic compositions produced by the methods of theinvention is measured by serum IgG or opsonophagocytic antibodyGeometric Mean Titers. In one aspect, the enhanced immune response to apneumococcal serotype is 10% or greater compared topolysaccharide-protein conjugate from the same pneumococcal serotypeprepared under aqueous conditions. In one embodiment, the animal is amouse. In another embodiment, the animal is a human.

In certain embodiments, the methods are employed with a human subjectwhich is 50 years old or older. In certain embodiments, the methods areemployed with a human subject which is 2 years old or younger. Incertain embodiments, the methods are employed with a human subject whichis immunocompromised.

The invention also provides methods of preparing a pneumococcalpolysaccharide-protein conjugate by reductive amination, the methodcomprising:

a) reacting a Streptococcus pneumoniae polysaccharide selected fromserotypes 3, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F withan amount of an oxidant (e.g., a periodate) to form an activatedpolysaccharide having an activation level from 0.05 to 0.22;

b) reacting the activated polysaccharide with a carrier protein in anaprotic solvent, optionally in the presence of a reducing agent, to forma polysaccharide-protein conjugate;

wherein the conjugate has a lysine loss value between 7.0 to 18.0inclusive.

In certain embodiments, the activation level is from 0.09 to 0.22.

In certain embodiments, the oxidant is periodate.

In certain embodiments, the activation level is measured by derivitizingaldehydes on the polysaccharide with thiosemicarbazide.

In certain embodiments, the reducing agent is a cyanoborohydride saltsuch as sodium cyanoborohydride.

In certain embodiments, the carrier protein is selected from the groupconsisting of tetanus toxoid, diphtheria toxoid, and CRM₁₉₇. In oneembodiment, the carrier protein is CRM₁₉₇.

The present invention also provides a quantitative method for determingthe aldehyde level (i.e., the level of periodate activation) in anactivated polysaccharide comprising the steps of:

a) derivatizing the activated polysaccharide to form a derivatizedpolysaccharide by reacting with a derivatizing agent until completion(i.e., the reaction plateaus);

b) isolating the derivatized polysaccharide by high performance sizeexclusion chromatography (to remove unreacted derivatizing agent andmatrix components);

c) quantifying the UV absorbance of the derivatized polysaccharide.

The derivatizing agent may be selected from the group consisting ofthiosemicarbazide, thiosemicarbazide structural analogs, hydrazides,hydrazine, semicarbazide, semicarbazide structural analogs, aminooxycompounds or aromatic amines.

In one embodiment, the quantifying in step c) is by comparison to aderivative standard. In one embodiment, the quantifying in step c) is bymeasurement against predetermined extinction coefficient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Extent of conjugation at different lysine sites on CRM₁₉₇ asdetermined by tryptic peptide mapping. Serotype 19A Ps-CRM₁₉₇conjugates, prepared by reductive amination in either aqueous solutionor DMSO, were digested by trypsin and analyzed by LC-UV-MS. The loss ofpeptide signal compared to CRM₁₉₇ control samples was plotted againstthe sites of conjugation.

FIG. 2. Electrochemiluminescent (ECL) immunogenicity results from mousestudy arms comparing serotype 3 Ps-CRM₁₉₇ conjugates prepared byreductive amination in either aqueous solution or DMSO. Conjugatesformulated with aluminum phosphate adjuvant (APA). Pre-vaccination (Pre)and post-dose 3 (PD3) results are shown. Results for APA only controlare also shown.

FIG. 3. Post-dose 3 opsophagocytic activity (OPA) results from mousestudy arms comparing serotype 3 Ps-CRM₁₉₇ conjugates prepared byreductive amination in either aqueous solution or DMSO. OPA resultspre-vaccination (Pre-immune) and for APA only control are also shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides immunogenic compositions comprisingpneumococcal polysaccharide-protein conjugates, in which conjugates fromat least one pneumococcal serotype are prepared using reductiveamination in an aprotic solvent such as DMSO. The present invention isbased, in part, on the discovery that the use of DMSO as a solventduring reductive amination of polysaccharide-protein conjugates resultsin the unexpectedly superior stability and enhanced immunogenicity forthose serotypes relative to the same conjugates prepared under aqueousconditions. The present invention relates to the advantages of DMSOsolvent in enhancing the covalent associations of polysaccharide toprotein through direct consumption of lysine residues on the surface ofthe carrier protein. For most serotypes tested, a “lysine loss” thatprovided good immunogenicity (≥7.0) could be achieved at a lowerpolysaccharide activation level (0.05 to 0.22) through conjugation in anaprotic solvent than in an aqueous buffer. The increased covalentassociation has a direct benefit to increasing the stability of thepolysaccharide protein conjugate and in enhancing the immune response tothose particular polysaccharide antigens conjugated in DMSO.

Without being bound by any theory, one possible mechanism for theenhanced immunogenicity observed with glycoconjugates prepared in DMSOinclude an increased number of linkages between the carbohydrate(capsular polysaccharide) and lysine residues on the surface of thecarrier protein which would result in additional attachment pointsbetween the protein and polysaccharide to impart stability and counterchemical depolymerization or breakdown of the peptide carbohydrate bond.See, e.g., Hsieh, Characterization of Saccharide-CRM₁₉₇ ConjugateVaccines in Brown F, Corbel M, Griffiths E (eds): Physico-ChemicalProcedures for the Characterization of Vaccines. Dev. Biol. Basel,Karger, 2000, vol 103, pp. 93-104. An additional benefit of theincreased polysaccharide-protein linkages that are created duringconjugation in the DMSO solvent could be additional opportunities forsuccessful presentation of peptide-carbohydrate to T-cells. It can beappreciated that due to the genetic variability in the human populationresulting in varying abilities and sensitivity of loading or associatingwith specific peptide sequences conjugated to carbohydrate antigens,that additional points of attachment on the carrier protein would allowfor increased chances for successful antigen presentation at the surfaceof an APC to allow for a T-cell dependent response to an otherwiseT-cell independent antigen. Another possible mechanism of enhancedimmunogenicity observed by conjugation in the DMSO solvent could be dueto the denaturation of CRM₁₉₇ in organic solvent, which exposesadditional lysines for polysaccharide linkages giving increased chancesfor glycopeptide presentation at the surface of an APC for T-celldependent response to different peptide epitopes. See Avci et al., 2011,Nature Medicine 17: 1602-1610.

Yet another benefit of conjugation in an organic solvent generatingdenatured CRM₁₉₇ in the conjugates could be reduced immunologicalinterference of antibodies against native CRM₁₉₇ epitopes. A furtherbenefit of the increased polysaccharide-protein linkages that arecreated during conjugation in the DMSO solvent could be the formation oflarger sized polysaccharide protein conjugates resulting in enhancedimmunogenicity. The compositions of the invention are believed toprovide significant advantages in eliciting a human response.

As shown in the Example 5, a polysaccharide protein conjugate preparedfrom S. pneumoniae serotype 3 using reductive amination in DMSO showedincreased immunogenicity (compared to the same conjugate prepared usingreductive amination in water) in a mouse model as measured byopsophagocytic activity (OPA). Moreover, as shown in the Example 6, a15-valent pneumococcal conjugate vaccine having seven serotypes preparedusing reductive amination in DMSO (and the other eight prepared in anaqueous solvent) tended to show superior immunogenicity in humans (with4 serotypes superior with statistical significance) for all sevenserotypes prepared in DMSO compared to the corresponding 15-valent PCVwhere all 15 seroytpes were prepared in an aqueous solvent.

As shown in Example 4, plotting the peptide signal decrease for lysinelocations on the CRM₁₉₇ protein in serotype 19A conjugates againstpossible sites of conjugation (compared to a CRM197 control) uncoveredadditional conjugation sites located in previously identified commonhuman T-cell peptide epitopes (See Raju et al., 1995, Eur. J. Immunol.25: 3207-3214, located in peptide 411-430 and peptide 431-450 of CRM₁₉₇sequence). Accordingly, in certain embodiments, the present invention isalso directed to immunogenic compositions comprising one or morepolysaccharide-CRM₁₉₇ conjugates, wherein at least one of thepolysaccharide-CRM₁₉₇ conjugates is prepared in an aprotic solvent andwherein a conjugate prepared in an aprotic solvent demonstrates greateraccessibility of lysines residues within amino acids 411-430 or 431-450of CRM₁₉₇ compared to the same conjugate prepared in an aqeuous solvent.In certain embodiments, the present invention is also directed toimmunogenic compositions comprising one or more polysaccharide-CRM₁₉₇conjugates, wherein at least one of the polysaccharide-CRM₁₉₇ conjugatesis prepared in an aprotic solvent and wherein one or more lysineresidues within amino acids 411-430 or 431-450 of CRM₁₉₇ in a conjugateprepared in an aprotic solvent are conjugated more than 10%. In certainembodiments, the present invention is directed to methods for increasingthe accessibility of lysine residues within CRM₁₉₇, particularly withinamino acids 411-430 or 431-450 of CRM₁₉₇, comprising conjugating apolysaccharide to CRM₁₉₇ in an aprotic solvent. In certain aspects ofthis embodiment, one or more lysine residues within amino acids 411-430or 431-450 of CRM₁₉₇ in a conjugate prepared in an aprotic solvent areconjugated more than 10%. In these embodiments, the polysaccharide canbe from any organism suitable for preparing an immunogenic composition.In certain aspects, the polysaccharide is from N. meningitides or S.pneumoniae. The polysaccharide may be from any serotype of theseorganisms.

As used herein, the terms “aqueous solvent” or “aqueous conditions” whenused with conjugation, such as reductive amination, refers use of wateras the solvent for the conjugation reaction. The water may containbuffers and other components except that no organic solvent is present.

As used herein, the terms “aprotic solvent”, when used with conjugation,such as reductive amination, refers use of a polar aprotic solvent, or acombination of polar aprotic solvents, as the solvent for theconjugation reaction. Examples of polar aprotic solvents include, butare not limited to, dimethylsulfoxide (DMSO), dimethylformamide (DMF),and hexamethylphosphoramide (HMPA). The aprotic solvent may have somewater present, for example, up to 1%, 2%, 5%, 10% or 20%.

As used herein, “DMSO solvent” and “DMSO conditions” are usedinterchangeably.

As used herein, the term “comprises” when used with the immunogeniccomposition of the invention refers to the inclusion of any othercomponents (subject to limitations of “consisting of” language for theantigen mixture), such as adjuvants and excipients. The term “consistingof” when used with the multivalent polysaccharide-protein conjugatemixture refers to a mixture having those particular S. pneumoniaepolysaccharide protein conjugates and no other S. pneumoniaepolysaccharide protein conjugates from a different serotype.

As used herein, “lysine loss” refers to the lysine consumption duringconjugation and is determined by the difference between the averagemeasured amount of lysine in the conjugate and the expected amount oflysine in the starting protein. Example 4 describes one method fordetermining “lysine loss”.

As used herein, the term “polysaccharide” is meant to include anyantigenic saccharide element (or antigenic unit) commonly used in theimmunologic and bacterial vaccine arts, including, but not limited to, a“saccharide”, an “oligosaccharide”, a “polysaccharide”, a“liposaccharide”, a “lipo-oligosaccharide (LOS)”, a “lipopolysaccharide(LPS)”, a “glycosylate”, a “glycoconjugate” and the like.

When referring to percentages of serotypes in the immunogeniccomposition being prepared under in an aprotic solvent (e.g., DMSO) andthe remaining polysaccharide protein conjugates being prepared underaqueous conditions, it is meant to simply refer to the number ofserotypes prepared in an aprotic solvent divided by the total number ofserotypes in the composition.

As used herein, all ranges, for example, pH, temperature, andconcentrations, are meant to be inclusive. For example, a pH range from5.0 to 9.0 is meant to include a pH of 5.0 and a pH of 9.0. Similarly, atemperature range from 4 to 25° C. is meant to include the outer limitsof the range, i.e., 4° C. and 25° C.

Polysaccharide

S. pneumonia capsular polysaccharides that can be prepared according tothe methods of the invention, i.e., reductive amination in an aproticsolvent, include, but are not limited to, serotypes: 1, 2, 3, 4, 5, 6A,6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C,16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A,31, 33F, 34, 35A, 35B, 35F, and 38. The polysaccharides may be used inthe form of oligosaccharides. These are conveniently formed byfragmentation of purified polysaccharide (e.g. by hydrolysis), whichwill usually be followed by purification of the fragments of the desiredsize.

In certain embodiments, one or more of serotypes 1, 2, 3, 4, 5, 6C, 6D,7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A,23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38 are prepared usingreductive amination in an aprotic solvent. In certain aspects,pneumococcal polysaccharides from one or more of serotypes 1, 3, 4, 5,9V, 11A, 12F, and 14 are prepared using reductive amination in anaprotic solvent. In certain aspects, pneumococcal polysaccharides fromone or more of serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F,19F, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38are prepared using reductive amination in an aprotic solvent. In certainaspects, pneumococcal polysaccharides from one or both of serotypes 3 or18C are conjugated to a carrier protein using reductive amination in anaprotic solvent. Polysaccharides from the other serotypes in amultivalent composition may be conjugated using reductive amination inan aprotic solvent or in an aqueous solvent. Polysaccharides from theother serotypes in a multivalent composition may also be conjugatedusing other chemistries which may be in an aprotic solvent or in anaqueous solvent.

Capsular polysaccharides from Streptococcus pneumoniae can be preparedby standard techniques known to those skilled in the art. For example,polysaccharides can be isolated from bacteria and may be sized to somedegree by known methods (see, e.g., European Patent Nos. EP497524 andEP497525); and preferably by microfluidisation accomplished using ahomogenizer or by chemical hydrolysis. In one embodiment, eachpneumococcal polysaccharide serotype is grown in a soy-based medium. Theindividual polysaccharides are then purified through standard stepsincluding centrifugation, precipitation, and ultra-filtration. See,e.g., U.S. Patent Application Publication No. 2008/0286838 and U.S. Pat.No. 5,847,112. Polysaccharides can be sized in order to reduce viscosityin polysaccharide samples and/or to improve filterability for conjugatedproducts using techniques such as mechanical or chemical sizing.Chemical hydrolysis maybe conducted using acetic acid. Mechanical sizingmaybe conducted using High Pressure Homogenization Shearing.

In some embodiments, the purified polysaccharides before conjugationhave a molecular weight of between 5 kDa and 4,000 kDa. Molecular weightcan be calculated by size exclusion chromatography (SEC) combined withmultiangle light scattering detector (MALS) and refractive indexdetector (RI). In other such embodiments, the polysaccharide has amolecular weight of between 10 kDa and 4,000 kDa; between 50 kDa and4,000 kDa; between 50 kDa and 3,000 kDa; between 50 kDa and 2,000 kDa;between 50 kDa and 1 ,500 kDa; between 50 kDa and 1 ,000 kDa; between 50kDa and 750 kDa; between 50 kDa and 500 kDa;

between 100 kDa and 4,000 kDa; between 100 kDa and 3,000 kDa; 100 kDaand 2,000 kDa; between 100 kDa and 1 ,500 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa;between 100 and 400 kDa; between 200 kDa and 4,000 kDa; between 200 kDaand 3,000 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,500kDa; between 200 kDa and 1 ,000 kDa; or between 200 kDa and 500 kDa.

The purified polysaccharides can be chemically activated to make thesaccharides capable of reacting with the carrier protein. The purifiedpolysaccharides can be connected to a linker. Once activated orconnected to a linker, each capsular polysaccharide is separatelyconjugated to a carrier protein to form a glycoconjugate. Thepolysaccharide conjugates may be prepared by known coupling techniques.

The polysaccharide can be coupled to a linker to form apolysaccharide-linker intermediate in which the free terminus of thelinker is an ester group. The linker is therefore one in which at leastone terminus is an ester group. The other terminus is selected so thatit can react with the polysaccharide to form the polysaccharide-linkerintermediate.

The polysaccharide can be coupled to a linker using a primary aminegroup in the polysaccharide. In this case, the linker typically has anester group at both termini. This allows the coupling to take place byreacting one of the ester groups with the primary amine group in thepolysaccharide by nucleophilic acyl substitution. The reaction resultsin a polysaccharide-linker intermediate in which the polysaccharide iscoupled to the linker via an amide linkage. The linker is therefore abifunctional linker that provides a first ester group for reacting withthe primary amine group in the polysaccharide and a second ester groupfor reacting with the primary amine group in the carrier molecule. Atypical linker is adipic acid N-hydroxysuccinimide diester (SIDEA).

The coupling can also take place indirectly, i.e. with an additionallinker that is used to derivatise the polysaccharide prior to couplingto the linker.

The polysaccharide is coupled to the additional linker using a carbonylgroup at the reducing terminus of the polysaccharide. This couplingcomprises two steps: (a1) reacting the carbonyl group with theadditional linker; and (a2) reacting the free terminus of the additionallinker with the linker. In these embodiments, the additional linkertypically has a primary amine group at both termini, thereby allowingstep (a1) to take place by reacting one of the primary amine groups withthe carbonyl group in the polysaccharide by reductive amination. Aprimary amine group is used that is reactive with the carbonyl group inthe polysaccharide. Hydrazide or hydroxylamino groups are suitable. Thesame primary amine group is typically present at both termini of theadditional linker. The reaction results in a polysaccharide-additionallinker intermediate in which the polysaccharide is coupled to theadditional linker via a C—N linkage.

The polysaccharide can be coupled to the additional linker using adifferent group in the polysaccharide, particularly a carboxyl group.This coupling comprises two steps: (a1) reacting the group with theadditional linker; and (a2) reacting the free terminus of the additionallinker with the linker. In this case, the additional linker typicallyhas a primary amine group at both termini, thereby allowing step (a1) totake place by reacting one of the primary amine groups with the carboxylgroup in the polysaccharide by EDAC activation. A primary amine group isused that is reactive with the EDAC-activated carboxyl group in thepolysaccharide. A hydrazide group is suitable. The same primary aminegroup is typically present at both termini of the additional linker. Thereaction results in a polysaccharide-additional linker intermediate inwhich the polysaccharide is coupled to the additional linker via anamide linkage.

In one embodiment, the chemical activation of the polysaccharides andsubsequent conjugation to the carrier protein by reductive amination canbe achieved by means described in U.S. Pat. Nos. 4,365,170, 4,673,574and 4,902,506, U.S. Patent Application Publication Nos. 2006/0228380,2007/184072, 2007/0231340 and 2007/0184071, and International PatentApplication Publication Nos. WO2006/110381, WO2008/079653, andWO2008/143709). The chemistry may entail the activation of pneumococcalpolysaccharide by reaction with any oxidizing agent which oxidizes aterminal hydroxyl group to an aldehyde, such as periodate (includingsodium periodate, potassium periodate, or periodic acid). The reactionleads to a random oxidative cleavage of vicinal hydroxyl groups of thecarbohydrates with the formation of reactive aldehyde groups.

In one embodiment, the polysaccharide is reacted with 0.01 to 10.0, 0.05to 5.0, 0.1 to 1.0, 0.5 to 1.0, 0.7 to 0.8, 0.05 to 0.5, 0.1 to 0.3molar equivalents of oxidizing agent. In a embodiment, thepolysaccharide is reacted with about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35,0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 molarequivalents of oxidizing agent. It is generally preferable to use loweramounts of periodate for activation, for example, 0.1 to 0.3 Meq, inorder to achieve limited polysaccharide activation (e.g., 0.05 to 0.22or 0.09 to 0.22 moles of aldehyde/mole of polysaccharide repeat unit).As used herein, “activation level” refers to moles of aldehyde/mole ofpolysaccharide repeat unit. Less polysaccharide activation results in amore native like polysaccharide, i.e., fewer hydroxyl groups areconverted to aldehydes.

In another embodiment, the duration of the oxidation reaction is between1 hour and 50 hours, between 10 hours and 30 hours, between 15 hours and20 hours, between 15 hours and 17 hours or about 16 hours.

In another embodiment, the temperature of the oxidation reaction ismaintained between 15° C. and 45° C., between 15° C. and 30° C., between20° C. and 25° C. In another embodiment, the temperature of the reactionis maintained at about 23° C.

Coupling to the carrier protein is by reductive amination via directamination to the lysyl groups of the protein. For example, conjugationis carried out by reacting a mixture of the activated polysaccharide andcarrier protein with a reducing agent such as sodium cyanoborohydride inthe presence of nickel. The conjugation reaction may take place underaqueous solution or in the presence of dimethylsulfoxide (DMSO). See,e.g., U.S. Patent Application Publication Nos. US2015/0231270 andUS2011/0195086 and European Patent No. EP 0471 177 B1. Unreactedaldehydes are then capped with the addition of a strong reducing agent,such as sodium borohydride.

Reductive amination involves two steps, (1) oxidation of thepolysaccharide to form reactive aldehydes, and (2) reduction of theimine (Schiff base) formed between activated polysaccharide and acarrier protein to form a stable amine conjugate bond. Before oxidation,the polysaccharide is optionally size reduced. Mechanical methods (e.g.homogenization) or chemical hydrolysis may be employed. Chemicalhydrolysis maybe conducted using acetic acid. The oxidation step mayinvolve reaction with periodate. For the purpose of the presentinvention, the term “periodate” includes both periodate and periodicacid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁻) and includes the various salts of periodate (e.g., sodium periodate and potassium periodate). In an embodiment, thecapsular polysaccharide is oxidized in the presence of metaperiodate,preferably in the presence of sodium periodate (NaIO₄). In anotherembodiment the capsular polysaccharide is oxidized in the presence oforthoperiodate, preferably in the presence of periodic acid.

In an embodiment, the oxidizing agent is a stable nitroxyl or nitroxideradical compound, such as piperidine-N-oxy or pyrrolidine-N-oxycompounds, in the presence of an oxidant to selectively oxidize primaryhydroxyls (as described in WO 2014/097099). In said reaction, the actualoxidant is the N-oxoammonium salt, in a catalytic cycle. In an aspect,said stable nitroxyl or nitroxide radical compound are piperidine-N-oxyor pyrrolidine-N-oxy compounds. In an aspect, said stable nitroxyl ornitroxide radical compound bears a TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) or a PROXYL(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In an aspect, saidstable nitroxyl radical compound is TEMPO or a derivative thereof. In anaspect, said oxidant is a molecule bearing a N-halo moiety. In anaspect, said oxidant is selected from the group consisting ofN-ChloroSuccinimide, N-Bromosuccinimide, N-lodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.

In certain aspects, the oxidizing agent is2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) free radical andN-Chlorosuccinimide (NCS) as the cooxidant (as described inInternational Patent Application Publication No. WO2014/097099).Therefore in one aspect, the glycoconjugates from S. pneumoniae areobtained by a method comprising the steps of: a) reacting a saccharidewith 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups (said method is designated“TEMPO/NCS-reductive amination” thereafter).

Optionally the oxidation reaction is quenched by addition of a quenchingagent. The quenching agent maybe selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid (such as glycerol, ethylene glycol, propan-1,2-diol,butan-1,2-diol or butan-2,3-diol, ascorbic acid).

The second step of the conjugation process is the reduction of the imine(Schiff base) bond between activated polysaccharide and a carrierprotein to form a stable conjugate bond (so-called reductive amination),using a reducing agent. Reducing agents which are suitable include thecyanoborohydrides (such as sodium cyanoborohydride or sodiumborohydride). In one embodiment the reducing agent is sodiumcyanoborohydride.

In certain embodiments of the methods of the invention, the reductiveamination reaction is carried out in aprotic solvent (or a mixture ofaprotic solvents). In an embodiment, the reduction reaction is carriedout in DMSO or in DMF (dimethylformamide) solvent. The DMSO or DMFsolvent may be used to reconstitute the activated polysaccharide andcarrier protein, if lyophilized. In one embodiment, the aprotic solventis DMSO.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, which may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH4). Suitable alternatives include sodiumtriacetoxyborohydride or sodium or zinc borohydride in the presence ofBronsted or Lewis acids, amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMe′PrN-BH₃, benzylamine-BH₃ or 5-ethyl-2-methylpyridine borane(PEMB) or borohydride exchange resin. Following the conjugation (thereduction reaction and optionally the capping), the glycoconjugates maybe purified (enriched with respect to the amount ofpolysaccharide-protein conjugate) by a variety of techniques known tothe skilled person. These techniques include dialysis,concentration/diafiltration operations, tangential flow filtration,precipitation/elution,column chromatography (ion exchangechromatography, multimodal ion exchange chromatography, DEAE, orhydrophobic interaction chromatography), and depth filtration. In anembodiment, the glycoconjugates are purified by diafilitration or ionexchange chromatography or size exclusion chromatography.

Glycoconjugates prepared using reductive amination in an aprotic solventare generally used in multivalent pneumococcal conjugate vaccines. Thus,in certain embodiments for multivalent compositions where not all theserotypes are prepared in an aprotic solvent, the reduction reaction forthe remaining seroytpes is carried out in aqueous solvent (e.g.,selected from PBS (phosphate buffered saline), MES(2-(N-morpholino)ethanesulfonic acid), HEPES,(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-tris, ADA(N-(2-Acetamido)iminodiacetic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO(3-Morpholino-2-hydroxypropanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), DIPSO (3-Bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), HEPPSO(N-(2-Hydroxyethyl)piperazine-N-(2-hydroxypropanesulfonic acid)), POPSO(Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA(triethanolamine), EPPS (4-(2-Hydroxyethyl)piperazine-1-propanesulfonicacid), Bicine or HEPB, at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0and 7.5).

In some embodiments, the glycoconjugates of the present inventioncomprise a polysaccharide having a molecular weight of between 10 kDaand 10,000 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 25 kDa and 5,000 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 50 kDaand 1,000 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 70 kDa and 900 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 100kDa and 800 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 200 kDa and 600 kDa. In further suchembodiments, the polysaccharide has a molecular weight of 100 kDa to1000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa;100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to 800 kDa;150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900 kDa;200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDa to500 kDa; 200 kDa to 400 kDa; 200 kDa to 300; 250 kDa to 1 ,000 kDa; 250kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to 600kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDato 1 ,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to 700kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDato 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700kDa; 400 kDa to 600 kDa; 500 kDa to 600 kDa.

In some embodiments, the glycoconjugates of the present invention have amolecular weight of between 1,000 kDa and 10,000 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 1,000kDa and 7,000 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 1,000 kDa and 6,000 kDa.

In certain embodiments, the conjugation reaction is performed byreductive amination wherein nickel is used for greater conjugationreaction efficiency and to aid in free cyanide removal. Transitionmetals are known form stable complexes with cyanide and are known toimprove reductive methylation of protein amino groups and formaldehydewith sodium cyanoborohydride (S Gidley et al., Biochem J. 1982, 203:331-334; Jentoft et al. Anal Biochem. 1980, 106: 186-190). By complexingresidual, inhibitory cyanide, the addition of nickel increases theconsumption of protein during the conjugation of and leads to formationof larger, potentially more immungenic conjugates.

Differences in starting cyanide levels in sodium cyanoborohydridereagent lots also lead to inconsistent conjugation performance,resulting in variable product attributes, such as conjugate size andconjugate Ps-to-CRM₁₉₇ ratio. The addition of nickel reduced conjugationinconsistency by complexing cyanide, eliminating differences in sodiumcyanoborohydride lots.

Suitable alternative chemistries include the activation of thesaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated saccharide may thus becoupled directly or via a spacer (linker) group to an amino group on thecarrier protein. For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide [e.g.ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB, or SIA,or SBAP). For example, the cyanate ester (optionally made by CDAPchemistry) is coupled with hexane diamine or adipic acid dihydrazide(ADH) and the amino-derivatised saccharide is conjugated to the carrierprotein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxylgroup on the protein carrier. Such conjugates are described inInternational Patent Application Publication Nos. WO 93/15760, WO95/08348 and WO 96/29094; and Chu et al., 1983, Infect. Immunity 40:245-256.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(See Bethell et al., 1979, J. Biol. Chem. 254: 2572-4; Hearn et al.,1981, J. Chromatogr. 218: 509-18) followed by reaction of with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

After conjugation of the capsular polysaccharide to the carrier protein,the polysaccharide-protein conjugates are purified (enriched withrespect to the amount of polysaccharide-protein conjugate) by one ormore of a variety of techniques. Examples of these techniques are wellknown to the skilled artisan and include concentration/diafiltrationoperations, ultrafiltration, precipitation/elution, columnchromatography, and depth filtration. See, e.g., U.S. Pat. No.6,146,902.

Another way to characterize the glycoconjugates of the invention is bythe number of lysine residues in the carrier protein (e.g., CRM₁₉₇) thatbecome conjugated to the saccharide which can be characterized as arange of conjugated lysines (degree of conjugation). The evidence forlysine modification of the carrier protein, due to covalent linkages tothe polysaccharides, can be obtained by amino acid analysis usingroutine methods known to those of skill in the art. Conjugation resultsin a reduction in the number of lysine residues recovered, compared tothe carrier protein starting material used to generate the conjugatematerials. In an embodiment, the degree of conjugation of theglycoconjugate of the invention is between 2 and 18, between 2 and 13,between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5,between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10,between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4,between 5 and 18, between 5 and 13, between 7 and 18, between 7, and 13,between 8 and 18, between 8 and 13, between 10 and 18 or between 10 and13. In an embodiment, the degree of conjugation of the glycoconjugate ofthe invention is about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 11 , about 12, about 13, about 14 orabout 15. In an embodiment, the degree of conjugation of theglycoconjugate of the invention is between 7 and 18. In some suchembodiments, the carrier protein is CRM₁₉₇.

The glycoconjugates of the invention may also be characterized by theratio (weight/weight) of saccharide to carrier protein. In someembodiments, the ratio of polysaccharide to carrier protein in theglycoconjugate (w/w) is between 0.5 and 3.0 (e.g., about 0.5, about 0.6,about 0.7, about 0.8, about 0.9, about 1.0, about 1.1 , about 1.2, about1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9,about 2.0, about 2.1 , about 2.2, about 2.3, about 2.4, about 2.5, about2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In otherembodiments, the saccharide to carrier protein ratio (w/w) is between0.5 and 2.0, between 0.5 and 1.5, between 0.8 and 1.2, between 0.5 and1.0, between 1.0 and 1.5 or between 1.0 and 2.0. In further embodiments,the saccharide to carrier protein ratio (w/w) is between 0.8 and 1.2. Inan embodiment, the ratio of capsular polysaccharide to carrier proteinin the conjugate is between 0.9 and 1.1. In some such embodiments, thecarrier protein is CRM₁₉₇. The glycoconjugates and immunogeniccompositions of the invention may contain free saccharide that is notcovalently conjugated to the carrier protein, but is neverthelesspresent in the glycoconjugate composition. The free saccharide may benon-covalently associated with (i.e., non-covalently bound to, adsorbedto, or entrapped in or with) the glycoconjugate.

In an embodiment, the glycoconjugate comprises less than about 50%, 45%,40%, 35%, 30%, 25%, 20% or 15% of free polysaccharide compared to thetotal amount of polysaccharide. In an embodiment the glycoconjugatecomprises less than about 25% of free polysaccharide compared to thetotal amount of polysaccharide. In an embodiment the glycoconjugatecomprises less than about 20% of free polysaccharide compared to thetotal amount of polysaccharide. In an embodiment the glycoconjugatecomprises less than about 15% of free polysaccharide compared to thetotal amount of polysaccharide.

Multivalent Polysaccharide-protein Conjugate Vaccines

Multivalent pneumococcal immunogenic compositions can comprise capsularpolysaccharides from S. pneumoniae serotype selected from at least oneof 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B,23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38 conjugated to oneor more carrier proteins, wherein a polysaccharide from at least oneserotype is prepared using reductive amination in an aprotic solventsuch as DMSO. The present invention contemplates multivalentpneumococcal immunogenic compositions having polysaccharides from atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 serotypes. Preferably, saccharides from a particularserotype are not conjugated to more than one carrier protein.

In certain embodiments, polysaccharides from at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 serotypesare prepared using reductive amination in an aprotic solvent such asDMSO.

In certain embodiments, one or more of serotypes 3, 6A, 6B, 7F, 18C,19A, 19F, or 23F are prepared using reductive amination in an aproticsolvent. In certain aspects of this embodiment, one or both of serotypes3 or 18C are prepared using reductive amination in an aprotic solvent.

In certain embodiments, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100% of the serotypes in a multivalent compositionare prepared in an aprotic solvent. The remainder of the serotypes areprepared using an alternative chemistry and/or in an aqueous solvent.

In certain embodiments, one or more of serotypes 1, 2, 3, 4, 5, 6C, 6D,7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A,23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38 are prepared usingreductive amination in an aprotic solvent. In certain aspects, one ormore of serotypes 1, 3, 4, 5, 9V, 11A, 12F, and 14 are prepared usingreductive amination in an aprotic solvent. In certain aspects, one ormore of serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 19F, 20,21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35B, 35F, and 38 are preparedusing reductive amination in an aprotic solvent.

In one embodiment, a multivalent composition consists of polysaccharidesfrom serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F prepared usingreductive amination in an aprotic solvent such as DMSO andpolysaccharides from serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F preparedusing reductive amination in an aqueous solvent.

After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention. Thesepneumococcal conjugates are prepared by separate processes and bulkformulated into a single dosage formulation.

Carrier Protein

In a particular embodiment of the present invention, CRM₁₉₇ is used asthe carrier protein. CRM₁₉₇ is a non-toxic variant (i.e., toxoid) ofdiphtheria toxin. In one embodiment, it is isolated from cultures ofCorynebacterium diphtheria strain C7 (β197) grown in casamino acids andyeast extract-based medium. In another embodiment, CRM₁₉₇ is preparedrecombinantly in accordance with the methods described in U.S. Pat. No.5,614,382. Typically, CRM₁₉₇ is purified through a combination ofultra-filtration, ammonium sulfate precipitation, and ion-exchangechromatography. In some embodiments, CRM₁₉₇ is prepared in Pseudomonasfluorescens using Pfenex Expression Technology™ (Pfenex Inc., San Diego,Calif.).

Other suitable carrier proteins include additional inactivated bacterialtoxins such as DT (Diphtheria toxoid) or fragment B of DT (DTFB), TT(tetanus toxid) or fragment C of TT, pertussis toxoid, cholera toxoid(e.g., as described in International Patent Application Publication No.WO 2004/083251), E. coli LT, E. coli ST, and exotoxin A from Pseudomonasaeruginosa. Bacterial outer membrane proteins such as outer membranecomplex c (OMPC), porins, transferrin binding proteins, pneumococcalsurface protein A (PspA; See International Application PatentPublication No. WO 02/091998), pneumococcal adhesin protein (PsaA), C5apeptidase from Group A or Group B streptococcus, or Haemophilusinfluenzae protein D, pneumococcal pneumolysin (Kuo et al., 1995, InfectImmun 63; 2706-13) including ply detoxified in some fashion for exampledPLY-GMBS (See International Patent Application Publication No. WO04/081515) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE andfusions of Pht proteins for example PhtDE fusions, PhtBE fusions (SeeInternational Patent Application Publication Nos. WO 01/98334 and WO03/54007), can also be used. Other proteins, such as ovalbumin, keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA) or purified proteinderivative of tuberculin (PPD), PorB (from N. meningitidis), PD(Haemophilus influenzae protein D; see, e.g., European Patent No. EP 0594 610 B), or immunologically functional equivalents thereof, syntheticpeptides (See European Patent Nos. EP0378881 and EP0427347), heat shockproteins (See International Patent Application Publication Nos. WO93/17712 and WO 94/03208), pertussis proteins (See International PatentApplication Publication No. WO 98/58668 and European Patent No.EP0471177), cytokines, lymphokines, growth factors or hormones (SeeInternational Patent Application Publication No. WO 91/01146),artificial proteins comprising multiple human CD4+ T cell epitopes fromvarious pathogen derived antigens (See Falugi et al., 2001, Eur JImmunol 31: 3816-3824) such as N19 protein (See Baraldoi et al., 2004,Infect Immun 72: 4884-7), iron uptake proteins (See International PatentApplication Publication No. WO 01/72337), toxin A or B of C. difficile(See International Patent Publication No. WO 00/61761), and flagellin(See Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used ascarrier proteins.

Other DT mutants can be used as the second carrier protein, such asCRM₁₇₆, CRM₂₂₈, CRM₄₅ (Uchida et al., 1973, J Biol Chem 218: 3838-3844);CRM₉, CRM₄₅, CRM₁₀₂, CRM₁₀₃ and CRM₁₀₇ and other mutations described byNicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, MaecelDekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Serand/or Ala 158 to Gly and other mutations disclosed in U.S. Pat. No.4,709,017 or U.S. Pat. No. 4,950,740; mutation of at least one or moreresidues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutationsdisclosed in U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; orfragment disclosed in U.S. Pat. No. 5,843,711. Such DT mutants can alsobe used to make DTFB variants where the variants comprise the B fragmentcontain the epitiope regions.

Where multivalent vaccines are used, a second carrier can be used forone or more of the antigens in a multivalent vaccine. The second carrierprotein is preferably a protein that is non-toxic and non-reactogenicand obtainable in sufficient amount and purity. The second carrierprotein is also conjugated or joined with an antigen, e.g., a S.pneumoniae polysaccharide to enhance immunogenicity of the antigen.Carrier proteins should be amenable to standard conjugation procedures.In one embodiment, each capsular polysaccharide not conjugated to thefirst carrier protein is conjugated to the same second carrier protein(e.g., each capsular polysaccharide molecule being conjugated to asingle carrier protein). In another embodiment, the capsularpolysaccharides not conjugated to the first carrier protein areconjugated to two or more carrier proteins (each capsular polysaccharidemolecule being conjugated to a single carrier protein). In suchembodiments, each capsular polysaccharide of the same serotype istypically conjugated to the same carrier protein.

Pharmaceutical/Vaccine Compositions

The present invention further provides compositions, includingpharmaceutical, immunogenic and vaccine compositions, comprising,consisting essentially of, or alternatively, consisting of any of thepolysaccharide serotype combinations described above together with apharmaceutically acceptable carrier and an adjuvant.

Formulation of the polysaccharide-protein conjugates of the presentinvention can be accomplished using art-recognized methods. Forinstance, individual pneumococcal conjugates can be formulated with aphysiologically acceptable vehicle to prepare the composition. Examplesof such vehicles include, but are not limited to, water, bufferedsaline, polyols (e.g., glycerol, propylene glycol, liquid polyethyleneglycol) and dextrose solutions.

In an embodiment, the vaccine composition is formulated in L-histidinebuffer with sodium chloride.

As defined herein, an “adjuvant” is a substance that serves to enhancethe immunogenicity of an immunogenic composition of the invention. Animmune adjuvant may enhance an immune response to an antigen that isweakly immunogenic when administered alone, e.g., inducing no or weakantibody titers or cell-mediated immune response, increase antibodytiters to the antigen, and/or lowers the dose of the antigen effectiveto achieve an immune response in the individual. Thus, adjuvants areoften given to boost the immune response and are well known to theskilled artisan. Suitable adjuvants to enhance effectiveness of thecomposition include, but are not limited to:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example, (a) MF59(International Patent Application Publication No. WO 90/14837),containing 5% Squalene, 0.5% TWEEN® 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalene, 0.4%TWEEN® 80, 5% pluronic-blocked polymer L121, and thr-MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, (c) Ribi™ adjuvant system (RAS), (Corixa,Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN® 80, and one or morebacterial cell wall components from the group consisting of3-O-deaylated monophosphorylipid A (MPL™) described in U.S. Pat. No.4,912,094, trehalose dimycolate (TDM), and cell wall skeleton (CWS),preferably MPL+CWS (Detox™); and (d) a Montanide ISA;

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (see, e.g., U.S. Pat. No. 5,057,540) may be used orparticles generated therefrom such as ISCOM (immunostimulating complexesformed by the combination of cholesterol, saponin, phospholipid, andamphipathic proteins) and Iscomatrix® (having essentially the samestructure as an ISCOM but without the protein);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529), which isformulated as an aqueous form or as a stable emulsion;

(5) synthetic polynucleotides such as oligonucleotides containing CpGmotif(s) (U.S. Pat. No. 6,207,646);

(6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules B7-1 and B7-2, etc; and

(7) complement, such as a trimer of complement component C3d.

In another embodiment, the adjuvant is a mixture of 2, 3, or more of theabove adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum salt. The aluminumsalt adjuvant may be an alum-precipitated vaccine or an alum-adsorbedvaccine. Aluminum-salt adjuvants are well known in the art and aredescribed, for example, in Harlow, E. and D. Lane (1988; Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992;Aluminum salts. Research in Immunology 143: 489-493). The aluminum saltincludes, but is not limited to, hydrated alumina, alumina hydrate,alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate,alhydrogel, SUPERFOS®, Amphogel, aluminum (III) hydroxide, aluminumhydroxyphosphate sulfate, Aluminum Phosphate Adjuvant (APA), amorphousalumina, trihydrated alumina, or trihydroxyaluminum.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA ismanufactured by blending aluminum chloride and sodium phosphate in a 1:1volumetric ratio to precipitate aluminum hydroxyphosphate. After theblending process, the material is size-reduced with a high-shear mixerto achieve a monodisperse particle size distribution. The product isthen diafiltered against physiological saline and steam sterilized.

In certain embodiments, a commercially available Al(OH)₃ (e.g.Alhydrogel or SUPERFOS® of Denmark/Accurate Chemical and Scientific Co.,Westbury, N.Y.) is used to adsorb proteins in a ratio of 50-200 gprotein/mg aluminum hydroxide. Adsorption of protein is dependent, inanother embodiment, on the pI (Isoelectric pH) of the protein and the pHof the medium. A protein with a lower pI adsorbs to the positivelycharged aluminum ion more strongly than a protein with a higher pI.Aluminum salts may establish a depot of antigen that is released slowlyover a period of 2-3 weeks, be involved in nonspecific activation ofmacrophages and complement activation, and/or stimulate innate immunemechanism (possibly through stimulation of uric acid). See, e.g.,Lambrecht et al., 2009, Curr Opin Immunol 21: 23.

Monovalent bulk aqueous conjugates are typically blended together anddiluted to target 8 μg/mL for all serotypes except 6B, which will bediluted to target 16 μg/mL. Once diluted, the batch will be filtersterilized, and an equal volume of aluminum phosphate adjuvant addedaseptically to target a final aluminum concentration of 250 μg/mL. Theadjuvanted, formulated batch will be filled into single-use, 0.5 mL/dosevials.

In certain embodiments, the adjuvant is a CpG-containing nucleotidesequence, for example, a CpG-containing oligonucleotide, in particular,a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment,the adjuvant is ODN 1826, which may be acquired from ColeyPharmaceutical Group.

“CpG-containing nucleotide,” “CpG-containing oligonucleotide,” “CpGoligonucleotide,” and similar terms refer to a nucleotide molecule of6-50 nucleotides in length that contains an unmethylated CpG moiety.See, e.g., Wang et al., 2003, Vaccine 21: 4297. In another embodiment,any other art-accepted definition of the terms is intended.CpG-containing oligonucleotides include modified oligonucleotides usingany synthetic internucleoside linkages, modified base and/or modifiedsugar.

Methods for use of CpG oligonucleotides are well known in the art andare described, for example, in Sur et al., 1999, J Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127: 139-58; and Yasuda etal., 2006, Crit Rev Ther Drug Carrier Syst. 23: 89-110.

Administration/Dosage

The compositions and formulations of the present invention can be usedto protect or treat a human susceptible to infection, e.g., apneumococcal infection, by means of administering the vaccine via asystemic or mucosal route. In one embodiment, the present inventionprovides a method of inducing an immune response to a S. pneumoniaecapsular polysaccharide conjugate, comprising administering to a humanan immunologically effective amount of an immunogenic composition of thepresent invention. In another embodiment, the present invention providesa method of vaccinating a human against a pneumococcal infection,comprising the step of administering to the human an immunogicallyeffective amount of a immunogenic composition of the present invention.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, in another embodiment, thedosage for human vaccination is determined by extrapolation from animalstudies to human data. In another embodiment, the dosage is determinedempirically. We have demonstrated that the vaccine is immunogenic inInfant Rhesus Monkey animal data.

“Effective amount” of a composition of the invention refers to a doserequired to elicit antibodies that significantly reduce the likelihoodor severity of infectivity of a microbe, e.g., S. pneumonia, during asubsequent challenge.

The methods of the invention can be used for the prevention and/orreduction of primary clinical syndromes caused by microbes, e.g., S.pneumonia, including both invasive infections (meningitis, pneumonia,and bacteremia), and noninvasive infections (acute otitis media, andsinusitis).

Administration of the compositions of the invention can include one ormore of: injection via the intramuscular, intraperitoneal, intradermalor subcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory or genitourinary tracts. In one embodiment,intranasal administration is used for the treatment of pneumonia orotitis media (as nasopharyngeal carriage of pneumococci can be moreeffectively prevented, thus attenuating infection at its earlieststage).

The amount of conjugate in each vaccine dose is selected as an amountthat induces an immunoprotective response without significant, adverseeffects. Such amount can vary depending upon the pneumococcal serotype.Generally, for polysaccharide-based conjugates, each dose will comprise0.1 to 100 μg of each polysaccharide, particularly 0.1 to 10 μg, andmore particularly 1 to 5 μg. For example, each dose can comprise 100,150, 200, 250, 300, 400, 500, or 750 ng or 1, 1.5, 2, 3, 4, 5, 6, 7,7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60,70, 80, 90, or 100 μg.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, in another embodiment, thedosage for human vaccination is determined by extrapolation from animalstudies to human data. In another embodiment, the dosage is determinedempirically.

In one embodiment, the dose of the aluminum salt is 10, 15, 20, 25, 30,50, 70, 100, 125, 150, 200, 300, 500, or 700 μg, or 1, 1.2, 1.5, 2, 3, 5mg or more. In yet another embodiment, the dose of alum salt describedabove is per μg of recombinant protein.

According to any of the methods of the present invention and in oneembodiment, the subject is human. In certain embodiments, the humansubject is an infant (less than 1 year of age), toddler (approximately12 to 24 months), or young child (approximately 2 to 5 years). In otherembodiments, the human subject is an elderly subject (e.g., >50 yearsold or >65 years old). The compositions of this invention are alsosuitable for use with older children, adolescents and adults (e.g., aged18 to 45 years or 18 to 65 years).

In one embodiment of the methods of the present invention, a compositionof the present invention is administered as a single inoculation. Inanother embodiment, the vaccine is administered twice, three times orfour times or more, adequately spaced apart. For example, thecomposition may be administered at 1, 2, 3, 4, 5, or 6 month intervalsor any combination thereof. The immunization schedule can follow thatdesignated for pneumococcal vaccines. For example, the routine schedulefor infants and toddlers against invasive disease caused by S.pneumoniae is 2, 4, 6 and 12-15 months of age. Thus, in an embodiment,the composition is administered as a 4-dose series at 2, 4, 6, and 12-15months of age.

The compositions of this invention may also include one or more proteinsfrom S. pneumoniae. Examples of S. pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationPublication Nos. WO 02/083855 and WO 02/053761.

Formulations

The compositions of the invention can be administered to a subject byone or more methods known to a person skilled in the art, such asparenterally, transmucosally, transdermally, intramuscularly,intravenously, intra-dermally, intra-nasally, subcutaneously,intra-peritonealy, and formulated accordingly.

In one embodiment, compositions of the present invention areadministered via epidermal injection, intramuscular injection,intravenous, intra-arterial, subcutaneous injection, orintra-respiratory mucosal injection of a liquid preparation. Liquidformulations for injection include solutions and the like.

The composition of the invention can be formulated as single dose vials,multi-dose vials or as pre-filled syringes.

In another embodiment, compositions of the present invention areadministered orally, and are thus formulated in a form suitable for oraladministration, i.e., as a solid or a liquid preparation. Solid oralformulations include tablets, capsules, pills, granules, pellets and thelike. Liquid oral formulations include solutions, suspensions,dispersions, emulsions, oils and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueousor non-aqueous solutions, suspensions, emulsions or oils. Examples ofnonaqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate.

Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Examples of oilsare those of animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, olive oil, sunflower oil, fish-liver oil, anothermarine oil, or a lipid from milk or eggs.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic.However it is often preferred that a pharmaceutical composition forinfusion or injection is essentially isotonic, when it is administrated.Hence, for storage the pharmaceutical composition may preferably beisotonic or hypertonic. If the pharmaceutical composition is hypertonicfor storage, it may be diluted to become an isotonic solution prior toadministration.

The isotonic agent may be an ionic isotonic agent such as a salt or anon-ionic isotonic agent such as a carbohydrate. Examples of ionicisotonic agents include but are not limited to NaCl, CaCl₂, KCl andMgCl₂. Examples of non-ionic isotonic agents include but are not limitedto mannitol, sorbitol and glycerol.

It is also preferred that at least one pharmaceutically acceptableadditive is a buffer. For some purposes, for example, when thepharmaceutical composition is meant for infusion or injection, it isoften desirable that the composition comprises a buffer, which iscapable of buffering a solution to a pH in the range of 4 to 10, such as5 to 9, for example 6 to 8.

The buffer may for example be selected from the group consisting ofTRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate,citrate, carbonate, glycinate, histidine, glycine, succinate andtriethanolamine buffer.

The buffer may furthermore for example be selected from USP compatiblebuffers for parenteral use, in particular, when the pharmaceuticalformulation is for parenteral use. For example the buffer may beselected from the group consisting of monobasic acids such as acetic,benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic,adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric,polybasic acids such as citric and phosphoric; and bases such asammonia, diethanolamine, glycine, triethanolamine, and TRIS.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, glycols such as propylene glycols orpolyethylene glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), andPoloxamer 188 (P188) are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, olive oil,sunflower oil, fish-liver oil, another marine oil, or a lipid from milkor eggs.

The formulations of the invention may also contain a surfactant.Surfactants include, but are not limited to: the polyoxyethylenesorbitan esters surfactants (commonly referred to as the Tweens),especially PS-20 and PS-80; copolymers of ethylene oxide (EO), propyleneoxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™tradename, such as linear EO/PO block copolymers; octoxynols, which canvary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, withoctoxynol-9 (TRITON™ X-100, or t-octylphenoxypolyethoxyethanol) being ofparticular interest; (octylphenoxy)polyethoxyethanol (IGEPALCA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin);nonylphenol ethoxylates, such as the Tergitol™ NP series;polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl andoleyl alcohols (known as BRIJ™ surfactants), such as triethyleneglycolmonolauryl ether (BRIJ™ 30); and sorbitan esters (commonly known as theSPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Apreferred surfactant for including in the emulsion is PS-80.

Mixtures of surfactants can be used, e.g. PS-80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (PS-80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (TRITON™ X-100) is also suitable.Another useful combination comprises laureth 9 plus a polyoxyethylenesorbitan ester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as PS-80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as TRITON™ X-100, or otherdetergents in the TRITON™ series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

In certain embodiments, the composition consists essentially ofhistidine (20 mM), saline (150 mM) and 0.02% PS-20 or 0.04% PS-80 at apH of 5.8 with 250 ug/mL of APA (Aluminum Phosphate Adjuvant). PS-20 canrange from 0.005% to 0.1% (w/v) with the presence of PS-20 or PS-80 informulation controlling aggregation during simulated manufacture and inshipping using primary packaging. Process consists of combining blend ofup to 24 serotypes in histidine, saline, and PS-20 or PS-80 thencombining this blended material with APA and saline with or withoutantimicrobial preservatives.

The choice of surfactant may need to be optimized for different drugproducts and drug substances. For multivalent vaccines having 15 or moreserotypes, PS-20 and P188 are preferred. The choice of chemistry used tomake conjugate can also play an important role in the stabilization ofthe formulation. In particular, when the conjugation reactions used toprepare different polysaccharide protein conjugates in a multivalentcomposition include both aqueous solvent and DMSO solvent, has foundthat particular surfactant systems provide significant differences instability. Improved stability of polysachharide protein conjugates wasseen with polysorbate 20 alone or with poloxamer 188 in combination witha polyol.

The exact mechanism of how a specific detergent protects abiotherapeutic is poorly understood and cannot be predicted a priori.Possible stabilization mechanisms include preferential hydration,preferential exclusion, air/liquid interface competition betweenbiotherapeutic and surface, surface tension, and/or direct associationof the detergent with the biotherpeutic to mask hydrophobic patcheswhich serve as seeds for aggregation.

Poloxamer may also be used in the compositions of the invention. Apoloxamer is a nonionic triblock copolymer composed of a centralhydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked bytwo hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).Poloxamers are also known by the tradename Pluronic®. Because thelengths of the polymer blocks can be customized, many differentpoloxamers exist that have slightly different properties. For thegeneric term “poloxamer”, these copolymers are commonly named with theletter “P” (for poloxamer) followed by three digits, the first twodigits×100 give the approximate molecular mass of the polyoxypropylenecore, and the last digit×10 gives the percentage polyoxyethylene content(e.g., P407=Poloxamer with a polyoxypropylene molecular mass of 4,000g/mol and a 70% polyoxyethylene content). For the Pluronic® tradename,coding of these copolymers starts with a letter to define its physicalform at room temperature (L=liquid, P=paste, F=flake (solid)) followedby two or three digits. The first digit (two digits in a three-digitnumber) in the numerical designation, multiplied by 300, indicates theapproximate molecular weight of the hydrophobe; and the last digit×10gives the percentage polyoxyethylene content (e.g., L61=Pluronic® with apolyoxypropylene molecular mass of 1,800 g/mol and a 10% polyoxyethylenecontent). See U.S. Pat. No. 3740421.

Examples of poloxamers have the general formula:

HO(C₂H₄O)_(a)(C₃H₆O)_(b)(C₂H₄O)_(a)H,

wherein a and b blocks have the following values:

Pluronic ® Poloxamer a b Molecular Weight L31 2 16 1100 (average) L351900 (average) L44NF 124 12 20 2090 to 2360 L64 2900 (average) L81 2800(average) L121 4400 (average) P123 20 70 5750 (average) F68NF 188 80 277680 to 9510 F87NF 237 64 37 6840 to 8830 F108NF 338 141 44 12700 to17400 F127NF 407 101 56 9840 to 14600Molecular weight units, as used herein, are in Dalton (Da) or g/mol.

Preferably, the poloxamer generally has a molecular weight in the rangefrom 1100 to 17,400 Da, from 7,500 to 15,000 Da, or from 7,500 to 10,000Da. The poloxamer can be selected from poloxamer 188 or poloxamer 407.The final concentration of the poloxamer in the formulations is from0.001% to 5% weight/volume, or 0.025% to 1% weight/volume. In certainaspects, the polyol is propylene glycol and is at final concentrationfrom 1% to 20% weight/volume. In certain aspects, the polyol ispolyethylene glycol 400 and is at final concentration from 1% to 20%weight/volume.

Suitable polyols for the formulations of the invention are polymericpolyols, particularly polyether diols including, but are not limited to,propylene glycol and polyethylene glycol, Polyethylene glycol monomethylethers. Propylene glycol is available in a range of molecular weights ofthe monomer from ˜425 to ˜2700. Polyethylene glycol and Polyethyleneglycol monomethyl ether is also available in a range of molecularweights ranging from ˜200 to ˜35000 including but not limited to PEG200,PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME3350 and PEG MME 4000. A preferred polyethylene glycol is polyethyleneglycol 400. The final concentration of the polyol in the formulations ofthe invention may be 1% to 20% weight/volume or 6% to 20% weight/volume.

The formulation also contains a pH-buffered saline solution. The buffermay, for example, be selected from the group consisting of TRIS,acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate,carbonate, glycinate, histidine, glycine, succinate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. Thebuffer is capable of buffering a solution to a pH in the range of 4 to10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspect of the invention, thebuffer selected from the group consisting of phosphate, succinate,histidine, MES, MOPS, HEPES, acetate or citrate. The buffer mayfurthermore, for example, be selected from USP compatible buffers forparenteral use, in particular, when the pharmaceutical formulation isfor parenteral use. The concentrations of buffer will range from 1 mM to50 mM or 5 mM to 50 mM. In certain aspects, the buffer is histidine at afinal concentration of 5 mM to 50 mM, or succinate at a finalconcentration of 1 mM to 10 mM. In certain aspects, the histidine is ata final concentration of 20 mM±2 mM.

While the saline solution (i.e., a solution containing NaCl) ispreferred, other salts suitable for formulation include but are notlimited to, CaCl₂, KCl and MgCl₂ and combinations thereof. Non-ionicisotonic agents including but not limited to sucrose, trehalose,mannitol, sorbitol and glycerol may be used in lieu of a salt. Suitablesalt ranges include, but not are limited to 25 mM to 500 mM or 40 mM to170 mM. In one aspect, the saline is NaCl, optionally present at aconcentration from 20 mM to 170 mM.

In an embodiment, the formulations comprise a L-histidine buffer withsodium chloride.

In certain embodiments of the formulations described herein, thepolysaccharide-protein conjugates comprise one or more pneumococcalpolysaccharides conjugated to a carrier protein. The carrier protein canbe selected from CRM₁₉₇, diphtheria toxin fragment B (DTFB), DTFBC8,Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussistoxoid, cholera toxoid, E. coli LT, E. coli ST, exotoxin A fromPseudomonas aeruginosa, and combinations thereof. In one aspect, all ofthe polysaccharide-protein conjugates are prepared using aqueouschemistry. In another aspect, one or more of the polysaccharide proteinconjugates are prepared using DMSO solvent. As an example, thepolysaccharide-protein conjugate formulation can be a 15-valentpneumococcal conjugate (15vPnC) formulation wherein polysaccharideprotein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F areprepared using DMSO solvent and polysaccharide protein conjugates fromserotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F are prepared using aqueoussolvent.

In another embodiment, the pharmaceutical composition is delivered in acontrolled release system. For example, the agent can be administeredusing intravenous infusion, a transdermal patch, liposomes, or othermodes of administration. In another embodiment, polymeric materials areused; e.g. in microspheres in or an implant.

The compositions of this invention may also include one or more proteinsfrom S. pneumoniae. Examples of S. pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationPublication Nos. WO 02/083855 and WO 02/053761.

Having described various embodiments of the invention with reference tothe accompanying description and drawings, it is to be understood thatthe invention is not limited to those precise embodiments, and thatvarious changes and modifications may be effected therein by one skilledin the art without departing from the scope or spirit of the inventionas defined in the appended claims.

The following examples illustrate, but do not limit the invention.

EXAMPLES Example 1: Preparation of S. pneumoniae CapsularPolysaccharides

Methods of culturing pneumococci are well known in the art. See, e.g.,Chase, 1967, Methods of Immunology and Immunochemistry 1: 52. Methods ofpreparing pneumococcal capsular polysaccharides are also well known inthe art. See, e.g., European Patent No. EP0497524. Isolates ofpneumococcal subtypes are available from the American Type CultureCollection (Manassas, Va.). The bacteria are identified as encapsulated,non-motile, Gram-positive, lancet-shaped diplococci that arealpha-hemolytic on blood-agar. Subtypes can be differentiated on thebasis of Quelling reaction using specific antisera. See, e.g., U.S. Pat.No. 5,847,112.

Cell banks representing each of the S. pneumococcus serotypes presentwere obtained from the Merck Culture Collection (Rahway, N.J.) in afrozen vial. A thawed seed culture was transferred to the seed fermentorcontaining a pre-sterilized growth media appropriate for S. pneumoniae.The culture was grown in the seed fermentor with temperature and pHcontrol. The entire volume of the seed fermentor was transferred to aproduction fermentor containing pre-sterilized growth media. Theproduction fermentation was the final cell growth stage of the process.Temperature, pH, and the agitation rate were controlled.

The fermentation process was terminated via the addition of aninactivating agent. After inactivation, the batch was transferred to theinactivation tank where it was held at controlled temperature andagitation. Cell debris was removed using a combination of centrifugationand filtration. The batch was ultrafiltered and diafiltered. The batchwas then subjected to solvent-based fractionations that removeimpurities and recover polysaccharide.

Example 2: Conjugation of Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,19A, 19F, 22F, 23F, and 33F to CRM₁₉₇ Using Reductive Amination inAqueous Solution

The different polysaccharide serotypes were individually conjugated topurified CRM₁₉₇ carrier protein using a common process flow.Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM₁₉₇ was then conjugatedto the activated polysaccharide utilizing NiCl₂ (2 mM) in the reactionmixture, and the resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to serotype-specific values in section below.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and all serotypes, except serotype 19A, were 0.45-micronfiltered. All serotypes, except serotype 19A, were homogenized to reducethe molecular mass of the polysaccharide. Serotype 19A was not sizereduced due to its relatively low starting size. Homogenization pressureand number of passes through the homogenizer were controlled toserotype-specific targets (150-1000 bar; 4-7 passes) to achieve aserotype-specific molecular mass. Size-reduced polysaccharide was0.2-micron filtered and then concentrated and diafiltered against waterusing a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to a serotype-specifictemperature (4-22° C.) and pH (4-5) with a sodium acetate buffer tominimize polysaccharide size reduction due to activation. For allserotypes (except serotype 4), polysaccharide activation was initiatedwith the addition of a 100 mM sodium metaperiodate solution. The amountof sodium metaperiodate added was serotype-specific, ranging fromapproximately 0.1 to 0.5 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit. The serotype-specific charge of sodiummetaperiodate was to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). For serotype4, prior to the sodium metaperiodate addition, the batch was incubatedat approximately 50° C. and pH 4.1 to partially deketalize thepolysaccharide.

For all serotypes, with the exception of serotypes 5 and 7F, theactivated product was diafiltered against 10 mM potassium phosphate, pH6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Serotypes 5 and 7F were diafiltered against 10 mM sodium acetate.Ultrafiltration for all serotypes was conducted at 2-8° C.

Polysaccharide Conjugation to CRM₁₉₇

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate, pH 6.0 or pH 7.0, depending on the serotype. Thebuffer pH selected was to improve the stability of activatedpolysaccharide during the conjugation reaction. Purified CRM₁₉₇,obtained through expression in Pseudomonas fluorescens as previouslydescribed (WO 2012/173876 A1), was 0.2-micron filtered and combined withthe buffered polysaccharide solution at a polysaccharide to CRM₁₉₇ massratio ranging from 0.4 to 1.0 w/w depending on the serotype. The massratio was selected to control the polysaccharide to CRM₁₉₇ ratio in theresulting conjugate. The polysaccharide and phosphate concentrationswere serotype-specific, ranging from 3.6 to 10.0 g/L and 100 to 150 mM,respectively, depending on the serotype. The serotype-specificpolysaccharide concentration was selected to control the size of theresulting conjugate. The solution was then 0.2-micron filtered. Nickelchloride was added to approximately 2 mM using a 100 mM nickel chloridesolution. Sodium cyanoborohydride (2 moles per mole of polysacchariderepeating unit) was added. Conjugation proceeded for a serotype-specificduration (72 to 120 hours) to maximize consumption of polysaccharide andprotein.

Reduction With Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. All serotypes (except serotype 5) werediafiltered against 100 mM potassium phosphate, pH 7.0 at 2-8° C. usinga 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch,recovered in the retentate, was then diluted to approximately 2.0 gpolysaccharide/L and pH-adjusted with the addition of 1.2 M sodiumbicarbonate, pH 9.4. Sodium borohydride (1 mole per mole ofpolysaccharide repeating unit) was added. 1.5 M potassium phosphate, pH6.0 was later added. Serotype 5 was diafiltered against 300 mM potassiumphosphate using a 100 kDa NMWCO tangential flow ultrafiltrationmembrane.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. The retentate batch was0.2 micron filtered.

Serotype 19F conjugate was incubated for approximately 7 days at 22° C.,diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0at 4° C. using a 100 kDa NMWCO tangential flow ultrafiltration membrane,and 0.2-micron filtered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0. Thebatch was dispensed into aliquots and frozen at ≤−60° C.

Example 3: Methods for the Conjugation of Serotypes 3, 4, 6A, 6B, 7F,9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F to CRM₁₉₇ Using ReductiveAmination in Dimethylsulfoxide

The different polysaccharide serotypes 3, 4, 6A, 6B, 7F, 9V, 14, 18C,19A, 19F, 22F, 23F, and 33F were individually conjugated to the purifiedCRM₁₉₇ carrier protein using a common process flow. Polysaccharide wasdissolved, sized to a target molecular mass, chemically activated andbuffer-exchanged by ultrafiltration. Activated polysaccharide andpurified CRM₁₉₇ were individually lyophilized and redissolved indimethylsuloxide (DMSO). Redissolved polysaccharide and CRM₁₉₇ solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled toserotype-specific values in section below.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water, and allserotypes, except serotype 19A, were 0.45-micron filtered. Allserotypes, except serotypes 18C and 19A, were homogenized to reduce themolecular mass of the Ps. Homogenization pressure and number of passesthrough the homogenizer were controlled to serotype-specific targets(150-1000 bar; 4-7 passes). Serotype 18C was size-reduced by acidhydrolysis at ≥90° C.

Size-reduced polysaccharide was 0.2-micron filtered and thenconcentrated and diafiltered against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane wasused for serotype 18C.

The polysaccharide solution was then adjusted to a serotype-specifictemperature (4-22° C.) and pH (4-5) with a sodium acetate buffer tominimize polysaccharide size reduction due to activation. For allserotypes (except serotype 4), polysaccharide activation was initiatedwith the addition of a 100 mM sodium metaperiodate solution. The amountof sodium metaperiodate added was serotype-specific, ranging fromapproximately 0.1 to 0.5 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit. The serotype-specific charge of sodiummetaperiodate was to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). For serotype4, prior to the sodium metaperiodate addition, the batch was incubatedat approximately 50° C. and pH 4.1 to partially deketalize thepolysaccharide.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane,then diafiltered or dialyzed against water using a 10 kDa NMWCOmembrane. A 5 kDa NMWCO membrane was used for serotype 18C.Ultrafiltration or dialysis for all serotypes was conducted at 2-8° C.

Polysaccharide Conjugation to CRM₁₉₇

Purified CRM₁₉₇, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2-5mM phosphate, pH 7.0 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

For serotypes other than serotype 3, the oxidized polysaccharides wereformulated at 6 mg Ps/mL and 5% w/v sucrose (50 mg sucrose/mL) in water.For serotype 3, the oxidized polysaccharide was formulated at 2 mg Ps/mLand 10% w/v sucrose (100 mg sucrose/mL) in water. The protein solutionwas formulated at 6 mg Pr/mL with 1% w/v sucrose (10 mg sucrose/mL) inphosphate buffer.

Formulated Ps and CRM₁₉₇ solutions were individually lyophilized.Lyophilized Ps and CRM₁₉₇ materials were redissolved in DMSO andcombined using a mixing tee. Sodium cyanoborohydride (1 moles per moleof polysaccharide repeating unit) was added, and conjugation proceededfor a serotype-specific duration (1 to 48 hours) to achieve a targetedconjugate size.

Reduction With Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction. The batch was diluted into150 mM sodium chloride, with or without approximately 0.025% (w/v)polysorbate 20, at approximately 4° C. Potassium phosphate buffer wasthen added to neutralize the pH. For serotypes 3, 6A, 6B, 7F, 9V, 18C,19A, 19F, 22F, 23F, and 33F, the batch was concentrated and diafilteredat approximately 4° C. against 150 mM sodium chloride, with or without25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flowultrafiltration membrane.

Final Filtration and Product Storage

Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 22F, 23F, and 33F wereconcentrated and diaftiltered against 10 mM histidine in 150 mM sodiumchloride, pH 7.0, with or without 0.015% (w/v) polysorbate 20, at 4° C.using a 300 kDa NMWCO tangential flow ultrafiltration membrane. Theretentate batch was 0.2 micron filtered.

Serotype 19F was incubated for approximately 5 days, diafiltered against10 mM histidine in 150 mM sodium chloride, pH 7.0 at approximately 4° C.using a 300 kDa NMWCO tangential flow ultrafiltration membrane, and0.2-micron filtered.

Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 19F, 22F, 23F, and 33F werediluted with additional 10 mM histidine in 150 mM sodium chloride, pH7.0, dispensed into aliquots and frozen at ≤-60° C.

Serotypes 4 and 14 were dialyzed against 150 mM sodium chloride atapproximately 4° C. using a 300 kDa NMWCO membrane, 0.2-micron filtered,dispensed into aliquots and frozen at ≤−60° C.

Example 4: Analysis of Conjugates Molecular Weight and Concentrationanalysis Of Conjugates Using HPSEC/UV/MALS/RI Assay

Conjugate samples were injected and separated by high performancesize-exclusion chromatography (HPSEC). Detection was accomplished withultraviolet (UV), multi-angle light scattering (MALS) and refractiveindex (RI) detectors in series. Protein concentration was calculatedfrom UV280 using an extinction coefficient. Polysaccharide concentrationwas deconvoluted from the RI signal (contributed by both protein andpolysaccharide) using the do/dc factors which are the change in asolution's refractive index with a change in the solute concentrationreported in mL/g. Average molecular weight of the samples werecalculated by Astra software (Wyatt Technology Corporation, SantaBarbara, Calif.) using the measured concentration and light scatteringinformation across the entire sample peak.

Polysaccharide Degree of Activation Assay

Conjugation occurs through reductive amination between the activatedaldehydes and mainly lysine residues on the carrier protein. The levelof activation, as represented by moles of aldehyde per moles ofpolysaccharide repeat unit, is important to control the conjugationreactions. An assay for measure the degree of activation is described inU.S. Patent Application Publication No. 2017/0021006.

An internal assay was developed to measure the degree of activationbased on reaction of aldehyde groups (created during periodate oxidationof the polysaccharide) with thiosemicarbazide (available from commercialsources).

Quantification can be achieved by NMR (nuclear magnetic resonance) or bycomparing the derivatized polysaccharide to appropriate referencestandards and/or through the use of extinction coefficients of thederivative. The use of extinction coefficients for this assay is similarto its use in HPSEC/UV/MALS/RI method.

Generally, the assay can be run under the following for reactionconditions:

Time: 0.5 h-35 hr (this is serotype specific, but the reaction isfollowed until completion, i.e., plateaus in a time course)

Temperature: 15° C.-37° C., preferably around 21-27° C.

TSC concentration: 1-5 mg/mL

pH of reaction: pH 3-5.5, preferably 4.0

For Example 4, polysaccharide was derivatized with 1.25 to 2.5 mg/mLthiosemicarbazide (TSC) at pH 4.0 to introduce a chromophore(derivatization of activated polysaccharide for serotypes 1, 5, and 9Vuses 1.25 mg/mL TSC). The derivatization reaction was allowed to proceedto reach a plateau. The actual time varied depending on reaction speedfor each serotype. TSC-Ps was then separated from TSC and other lowmolecular weight components by high performance size exclusionchromatography. The signal was detected by UV absorbance at 266 nm. Thelevel of activated aldehyde is calculated against either standard curveinjections of Mono-TSC or directly using predetermined extinctioncoefficients. Mono-TSC is a synthesized thiosemicarbazone derivative ofmonosaccharide. The aldehyde level is then converted to moles ofaldehyde per mole of repeat unit (Ald/RU) using the Ps concentrationmeasured by HPSEC/UV/MALS/RI assay.

Similar derivatization can be conducted with thiosemicarbazidestructural analogs, hydrazides, hydrazine, semicarbazide, semicarbazidestructural analogs, aminooxy compounds or aromatic amines as long as thederivatives have significant UV absorbance. The UV absorbance could befrom the chromophore attached to the derivatization agents, or achromophore generated as a result of the aldehyde derivatization, as thecase of thiosemicarbazide.

Determination of Lysine Consumption in Conjugated Protein as a Measureof the Number of Covalent Attachments Between Polysaccharide And CarrierProtein

The Waters AccQ-Tag amino acid analysis (AAA) was used to measure theextent of conjugation in conjugate samples. Samples were hydrolyzedusing vapor phase acid hydrolysis in the Eldex workstation, to break thecarrier proteins down into their component amino acids. The free aminoacids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC). The derivatized samples were then analyzed using UPLCwith UV detection on a C18 column. The average protein concentration wasobtained using representative amino acids other than lysine. Lysineconsumption during conjugation (i.e., lysine loss) was determined by thedifference between the average measured amount of lysine in theconjugate and the expected amount of lysine in the starting protein.

Attributes of Conjugates Made Using Reductive Amination in Aqueous andDMSO Solutions

Polysaccharide activation and lysine consumption (i.e., lysine loss)results for conjugates generated using the processes described inExamples 2 and 3 are listed in Table 1. There is a clear distinctionthat conjugates made in DMSO (Example 3) had higher lysine consumptionwith lower polysaccharide activation than conjugates made in aqueoussolution (Example 2). This suggests that preparing the conjugates inDMSO solution allows the polysaccharide to attach to more conjugationsites on the carrier protein with less activation or destruction tonative polysaccharide structures. As a result, the conjugates on averagecontain more glycopeptide per polysaccharide repeating unit due tohigher cross-linking in conjugates prepared in DMSO solution than inaqueous solution. It is believed that the glycopeptide is the antigenicdomain to which an immune response is generated. Consequently,conjugates generated in DMSO are expected to be more immunogenic thatconjugates generate in aqueous solution.

The average molecular weight (Mw) of the conjugates in Table 1 weremeasured by the HPSEC UV-MALS-RI assay. Conjugates generated byreductive amination in aqueous solution ranged from 990 to 3410 kDa.Conjugates generated in DMSO were generally larger with sizes rangingfrom 1300 to 5822 kDa.

TABLE 1 Lysine loss for pneumococcal serotype 3, 4, 6A, 6B, 7F, 9V, 14,18C, 19A, 19F, and 23F CRM₁₉₇ conjugates made using reductive aminationin aqueous solution or in DMSO. Conjugation Polysaccharide reaction inactivation (mole Lysine loss Conjugate aqueous or aldehyde/mole (mol/molConjugate Lot # DMSO solution repeat unit) protein) Serotype 3- 1Aqueous 0.10 3.1 CRM₁₉₇ 2 0.10 2.5 3 0.10 3.1 4 DMSO 0.092 16.3 5 0.0539.6 Serotype 4- 1 Aqueous 0.43 2.7 CRM₁₉₇ 2 DMSO 0.25 3.0 Serotype 6A- 1Aqueous 0.19 4.5 CRM₁₉₇ 2 DMSO 0.11 9.1 Serotype 6B- 1 Aqueous 0.18 4.6CRM₁₉₇ 2 DMSO 0.11 9.6 Serotype 7F- 1 Aqueous 0.26 2.0 CRM₁₉₇ 2 DMSO0.22 10.6 Serotype 9V- 1 Aqueous 0.30 4.7 CRM₁₉₇ 2 DMSO 0.15 7.9Serotype 14- 1 Aqueous 0.22 6.4 CRM₁₉₇ 2 DMSO 0.22 12.7 Serotype 18C- 1Aqueous 0.12 3.5 CRM₁₉₇ 2 DMSO 0.11 9.2 Serotype 19A- 1 Aqueous 0.38 4.9CRM₁₉₇ 2 DMSO 0.14 9.5 Serotype 19F- 1 Aqueous 0.13 2.7 CRM₁₉₇ 2 DMSO0.15 9.6 Serotype 22F- 1 Aqueous 0.12 1.7 CRM₁₉₇ 2 DMSO 0.15 7.2 3 0.157.0 Serotype 23F- 1 Aqueous 0.39 3.2 CRM₁₉₇ 2 DMSO 0.19 10.8 Serotype33F- 1 Aqueous 0.23 4.5 CRM₁₉₇ 2 DMSO 0.14 7.0

Quantification of the Extent of Conjugation at Different Sites on CRM₁₉₇

Polysaccharide can be conjugated either to the amine group at theN-terminus of the carrier protein or to any of the side chains of the 39lysine residues in CRM₁₉₇. The amino acid sequence of CRM₁₉₇ is providedin Table 2, where the lysines (abbreviated as K) are underlined and inbold. To locate and quantify the extent of polysaccharide conjugation atthe different sites on CRM₁₉₇ protein, an LC/UV/MS peptide mappingmethod was used. Representative conjugate samples (prepared with DMSO oraqueous solution) were digested in duplicate with trypsin, producingtryptic peptides. The mixtures were then separated on a reversed phaseC18 column and analyzed by UV and mass spectrometer. A CRM₁₉₇ proteinsample (not conjugated with a polysaccharide) was also processed intriplicate at the same time as a control. Since trypsin cleaves aprotein on the C-terminal side of lysine and arginine residues,conjugation at a lysine residue makes that site protease resistant. Theextent of conjugation at a particular site was determined by calculatinga decrease of peak intensity of a tryptic peptide as compared to aCRM₁₉₇ control. Depending on the cleavage sites and sequences, thesignal decrease of a particular peptide could be due to mis-cleavage ofthe lysines at the preceding peptide, or mis-cleavage of the lysine atthe end of the peptide, or conjugation in the middle of the peptidesequence.

The relative percentages of peptide signal decrease for serotype 19Aconjugates compared to CRM₁₉₇ control were plotted against possiblesites of conjugation in FIG. 1. The lysine locations listed in thex-axis were numbered based on their order on the CRM₁₉₇ proteinsequence, and represent possible conjugation sites of the analyzedpeptides. For example, “33” means the peptide signal decrease was due toconjugation at the 33rd lysine; and “6, 7” means the peptide signaldecrease was due to conjugation at the 6th, or the 7th, or both lyines.The data in FIG. 1 suggested that not only the extent of conjugation ateach site was generally higher for conjugates prepared in DMSO comparedto aqueous solution, there were also more sites of conjugation in DMSO.Those additional conjugation sites include the 29th, 30th, 31st, and32nd lysines, which were only lysines located in previously identifiedcommon human T-cell peptide epitopes (See Raju et al., 1995, Eur. J.Immunol. 25: 3207-3214, located in peptide 411-430 and peptide 431-450of CRM₁₉₇ sequence). Similar results were observed with other serotypestested.

TABLE 2 CRM₁₉₇ amino acid sequence Amino Acid Amino Acid Sequence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 ID NO: 1,

Example 5: Mouse Immunogenicity Studies Comparing Serotype 3 Ps-CRM₁₉₇Conjugates Prepared in Aqueous Solution Versus DMSO

All animal experiments were performed in strict accordance with therecommendations in the Guide for Care and Use of Laboratory Animals ofthe National Institutes of Health. The protocol was approved by theInstitutional Animal Care and Use Committee (IACUC), MRL, West Point,Pa.

Eight week old female CD1 mice were housed in micro isolator cages(n=10/cage) in the animal facility at MRL, West Point, Pa. Food andwater were available ad libitum. Mice (n=10/group) were intramuscularly(IM) immunized with ST3-CRM₁₉₇ conjguates (0.4 μg ST3 polysaccharide),formulated with aluminum phosphate adjuvant (APA) as described in Table3. Negative control animals received APA alone. Immunizations wereperformed on days 0, 14 and 28. Blood was collected in serum separatortubes (BD, Franklin Lakes, N.J.) via tail vein on days 6 and 34.

TABLE 3 Mouse study arms comparing serotype 3 Ps-CRM₁₉₇ conjugatesprepared in aqueous solution versus DMSO solution. Arm # Arm Descriptionof Conjugate Description of Formulation 1 APA Control, no conjugate used250 μg/mL APA, 20 mM L- histidine, pH 5.8 and 150 mM NaCl with 0.2% w/vPS-20 2 ST3-CRM₁₉₇(aqueous)/ Monovalent ST3-CRM₁₉₇ 0.4 μg ST3-CRM₁₉₇,250 APA conjugate (Lot #1 in Table 1) μg/mL APA, 20 mM L- prepared byreductive histidine, pH 5.8 and 150 mM amination in aqueous solutionNaCl with 0.2% w/v PS-20 as described in Example 2 3 ST3-CRM₁₉₇(DMSO)/Monovalent ST3-CRM₁₉₇ 0.4 μg ST3-CRM₁₉₇, 250 APA conjugate (Lot #4 inTable 1) μg/mL APA, 20 mM L- prepared by reductive histidine, pH 5.8 and150 mM amination in DMSO as NaCl with 0.2% w/v PS-20 described inExample 3

Electrochemiluminescent (ECL) Immunogenicity Assays

Mouse antibody responses were measured in 96-well multiplexedelectrochemiluminescent assays as described previously with slightmodifications. See Marchese et al., 2009, Clin Vaccine Immunol 16(3):387-96; Skinner et al., 2011, Vaccine 29(48): 8870-6; and Caro-Aguilaret al., 2017 Vaccine 35(6): 865-72. Briefly, following test seraincubation for 1 hour on Meso-Scale Discovery plates (Meso ScaleDiagnostics, Rockville, Md.) and washing, 25 μl of a 2 μg/ml Sulfo-tag(Meso Scale Diagnostics, Rockville, Md.) labeled goat anti-mouse IgG wasadded to each well. Plates were incubated for 1 hour at room temperaturewhile shaking and then processed as described previously and read on aMESO Sector 5600.

The ECL titer was calculated as the reciprocal of the linearlyinterpolated dilution corresponding to the cutoff value (pneumococcalpolysaccharide ECL geometric mean signal of pre-determined positivecontrol pooled mouse sera). Interpolation was performed usinglogarithmic scaling for ECL and the dilution. Titer was then obtained byback-transforming the linearly interpolated dilution. Titers wereextrapolated for samples falling outside the studied dilution range of100 to 1,562,500, based on linear extrapolation (in the log-log scaling)using the intercept and slope of the last 3 ECL data points for thesample curve completely above the cutoff line or using the intercept andslope of the first 2 ECL data points for the sample curve completelybelow the cutoff line. Titer was then obtained by back-transforming thelinearly extrapolated dilution.

Opsonophagocytic Killing Assay (OPA)

Pneumococcal serotype 3 opsonophagocytosis killing assays (OPA) wereperformed as described previously with slight modifications(Caro-Aguilar et al., 2017 Vaccine 35(6): 865-72; and Burton et al.,2006, Clin Vaccine Immunol 13(9): 1004-9). Following incubation of thesera, bacteria, complement and HL-60 cells, 10 μl of theopsonophagocytic reaction was transferred to an individual well on aMillipore 96-well filter plate containing 200 μ/well of sterile water.The plate was vacuum filtered and 100 μl of Todd Hewett yeast extract(THYE, Teknova) broth was added. The medium was filtered and the moistplate was placed in a sealed plastic bag overnight at 27° C. Platefilters were then stained with 100 μl/well of a 0.1% Coomassie bluesolution (Bio-Rad, Hercules, Calif.). Stain was filtered through theplate, colonies were destained with Coomassie destaining solution(Bio-Rad) and vacuum filtered again until dry. Stained bacterialcolonies were counted on a CTL Immunospot reader (Shaker Heights, OH).The OPK titer was defined as the reciprocal of the serum dilution withat least 50% killing, compared to the average growth in the complementcontrol (no serum control) wells and was calculated by linearlyinterpolating between the consecutive dilutions whose signals bracket50% killing.

Results of pre-immunization and post dose 3 are illustrated in FIG. 2and Table 4 for ECL Immunogencity, and in FIG. 3 for OPA. Bothconjugates prepared by processes using aqueous and DMSO solutions areimmunogenic and provide functional killing activities against thebacteria. Interestingly, conjugate prepared by process using DMSOsolution gave both higher ECL immunogenicity and OPA responses thanconjugate prepared using aqueous solution. The ECL immunogenicitydifference is statistically significant. The GMT ratio of Arm 3 relativeto Arm 2 is 3.41 (with lower and upper 95% confidence interval of 1.26and 9.26).

TABLE 4 Post-Dose 3 ECL immunogenicity results of mouse study armscomparing serotype 3 Ps-CRM₁₉₇ conjugates prepared in aqueous solutionversus DMSO solution. Geometric Lower Upper Mean 95% 95% Arm Titer,confidence confidence # Arm GMT interval interval 1 APA 368 227 596 2ST3-CRM₁₉₇(aqueous)/ 355,207 187,905 671,466 APA 3 ST3-CRM₁₉₇(DMSO)/1,211,654 719,297 2,041,028 APA

Example 6: Adult Human Immunogenicity Studies Comparing PneumococcalPolysaccharide-Protein Conjugates Prepared with Reductive Amination inAqueous Solution Versus in DMSO

The immunogenicity and safety of two 15-valent pneumococcal conjugatevaccine (PCV15) in healthy Pneumococcal vaccine-naive adults 50 years ofage or older is described in this example.

Trial Design

A randomized, multi-site, double-blind trial was carried out to comparethe safety, tolerability and immunogenicity of a single dose of 2different PCV15 formulations (PCV15-A and PCV15-B) and Prevnar 13™(Pneumococcal 13-valent Conjugate Vaccine [Diphtheria CRM₁₉₇ Protein],Wyeth Pharmaceuticals Inc., a subsidiary of Pfizer Inc., Philadelphia,Pa., USA) in adult subjects 50 years of age or older in good health (anyunderlying chronic illness must be documented to be in stablecondition), to be conducted in conformance with Good Clinical Practices.

A total of 690 healthy Pneumococcal vaccine-naive individuals, 50 yearsof age or older, were enrolled, and randomized into three differentvaccination groups: Prevnar 13™, PCV15-A and PCV15-B with the ratio1:1:1. Randomization was stratified by age at study entry (50 to 64years, 65 to 74 years, and ≥75 years).

PCV15 contained 2 μg/0.5 mL dose of each of the following serotypes ofPneumococcal polysaccharide conjugated to CRM₁₉₇ (1, 3, 4, 5, 6A, 7F,9V, 14, 18C, 19A, 19F, 22F, 23F, 33F), 4 μg/0.5mL dose of serotype 6BPneumococcal polysaccharide conjugated to CRM₁₉₇, 125 μg/0.5mL dose ofAluminum Phosphate Adjuvant, 20 mM L-histidine, 150 mM Sodium Chloride,pH 5.8. PCV15-A was formulated with 0.2% w/v P188. PCV15-B wasformulated with 0.1% w/v PS-20.

For PCV15-A, all fifteen polysaccharide serotypes (1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) were conjugated to CRM₁₉₇using reductive amination in aqueous solution as described in Example 2.Attributes for some of these conjugates (Conjugate Lot #1 materials) arelisted in Table 1.

PCV15-B, serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F were conjugated toCRM₁₉₇ using reductive amination in DMSO described in Example 3.Attributes for these conjugates (Conjugate Lot #2 materials) are listedin Table 1. The conjugates for the remaining serotypes (1, 3, 4, 5, 9V,14, 22F, and 33F) are the same conjugates that were used in PCV15-A.

Both PCV15 formulations had generally comparable safety profiles toPrevnar 13™ based on the cumulative safety evaluation (data not shown).The serotype-specific IgG GMCs and OPA GMTs were measured at Day 30.(OPA results not included).

Results

The IgG Geometric Mean Concentrations (GMCs) and confidence intervals(CI) are summarized in the Table 6. Serotype 6A, 6B, 7F, 18C, 19A, 19F,and 23F conjugates in PCV15-A and PCV15-B were made with differentconjugation processes as described above. Consistent with the resultsshown in Table 4, the immunogenicity responses for each of the serotypesshown in Table 6 was improved when the polysaccharide serotypes wereconjugated to CRM₁₉₇ in DMSO. The GMCs for serotypes 18C, 19A, 19F, and23F in PCV15-B were significantly higher than those in PCV15-A (2-sidedalpha=0.05). These data strongly demonstrate the advantage ofconjugating in DMSO to improve immunogenicity. This discovery that hasnot been previously demonstrated for pneumococcal or other conjugatevaccines.

TABLE 6 Summary of IgG antibody responses of PCV15-A, and PCV15-Bformulation for serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F. Conjugatesof these serotypes were made using reductive amination in aqueoussolution (PCV15-A) or by reductive amination in DMSO (PCV15-B). PCV15-APCV15-B Estimated (N = 231), (N = 231), GMC Ratio^(†) GMC (Day 30)^(†)GMC^(†)(Day 30) [PCV15-B/ Estimated Estimated PCV15-A] Serotypes nResponse n Response (95% CI) ^(†) 6A 217 3.74 217 4.93 1.32 (0.96, 1.81)6B 217 3.69 217 4.95 1.34 (0.98, 1.85) 7F 217 4.09 217 4.53 1.11 (0.86,1.43) 18C 217 6.61 217 10.99 1.66 (1.27, 2.18) 19A 217 8.77 217 13.831.58 (1.23, 2.02) 19F 217 4.11 217 6.80 1.66 (1.26, 2.17) 23F 217 3.92217 5.53 1.41 (1.04, 1.91) ^(†)Estimated GMCs, GMC ratio, and 95% CI areobtained from a cLDA model. N = Number of subjects randomized andvaccinated. n = Number of subjects with Day 30 postvaccination serologyresults contributing to the analysis. GMC = Geometric MeanConcentration. CI = Confidence interval

1-44. (canceled)
 45. A method of preparing a pneumococcalpolysaccharide-protein conjugate by reductive amination, the methodcomprising: a) reacting a Streptococcus pneumoniae polysaccharideselected from serotypes 3, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F,and 33F with an amount of an oxidant to form an activated polysaccharidehaving an activation level from 0.05 to 0.22; and b) reacting theactivated polysaccharide with a carrier protein in an aprotic solvent toform a polysaccharide-protein conjugate; wherein the resultingpolysachharide-protein conjugate has a lysine loss value between 7.0 and18.0 inclusive.
 46. The method of claim 45, wherein the activation levelis from 0.09 to 0.22.
 47. The method of claim 45, wherein the reactingin step b) is in the presence of a reducing agent.
 48. The method ofclaim 45, wherein the carrier protein is selected from the groupconsisting of tetanus toxoid, diphtheria toxoid, and CRM₁₉₇.
 49. Themethod of claim 48, wherein the carrier protein is CRM₁₉₇. 50-53.(canceled)